Microchip capillary electrophoresis using on-line chemiluminescence detection

Chemiluminescence detection was used in capillary electrophoresis integrated on a microchip. Quartz microchips have two main channels and four reservoirs. Dansyl-lysine and -glycine were separated and detected with bis[(2-(3,6,9-trioxadecanyloxycarbony)-4-nitrophenyl]oxalate as peroxyoxalate chemiluminescent reagent. These dansyl amino acids came into contact with the chemiluminescence reagent to produce visible light at the interface between the separation channel and chemiluminescence reagent-containing reservoir. The detection limit (S/N = 3) for dansyl-lysine was 1 x 10(-5) M, which corresponded to the very small mass detection limit of ca. 0.4 fmol. However, the concentration sensitivity in the present system was approximately two orders of magnitude lower than that in the conventional capillary electrophoresis-chemiluminescence detection system. The relative standard deviations of migration time and peak height for dansyl-lysine were 4.2 and 4.5%, respectively. A channel conditioning before every run and an appropriate control of voltages were needed for the reproducible results. The present system had advantages in rapid separation time (within 40 s), small (several 10 pI) and accurate sample injection method using a cross-shaped injector, and simplification and miniaturization of the detection device.     

Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10⁻⁵ M luminol added to the CE running buffer and 5.0 × 10⁻⁵ M K₃[Fe(CN)₆] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10⁻⁸ to 2.5 × 10⁻⁶ M (r = 9991), and a detection limit of 2.0 × 10⁻⁸ M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10⁻⁷ M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.     

Determination of imazalil by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

A simple, rapid and reproducible capillary isotachophoretic on-line coupled with capillary zone electrophoresis (CITP-CZE) method for the determination of IMz in food packaging extracts and its residues in apples is described. A good separation of the IMZ from other sample constituents was achieved within 15 minutes without any sample clean up. Method characteristics (linearity, accuracy, intra-assay and detection limit) were determined. Less amount of time involved, sufficient sensitivity and low running cost are the important attributes of CITP-CZE method.     

Determination of clenbuterol by capillary electrophoresis immunoassay with chemiluminescence detection

A competitive immunoassay for clenbuterol (CLB) based on capillary electrophoresis with chemiluminescence (CL) detection was established. The method was based on the competitive reaction of horseradish peroxidase (HRP)-labeled CLB (CLB-HRP) and free CLB with anti-CLB antiserum. The factors affecting the electrophoresis and CL detection were systematically investigated with HRP as a model sample. Under the optimal conditions, the tracer CLB-HRP and the immunoassay complex were separated, and the linear range and the detection limit (S/N = 3) for CLB were 5.0-40 nmol l⁻¹ and 1.2 nmol l⁻¹, respectively. The proposed method has been applied satisfactorily in the analysis of urine sample.     

Determination of folic acid by capillary electrophoresis with chemiluminescence detection

A rapid and simple method is presented for the determination of folic acid (FA) by capillary electrophoresis (CE) with chemiluminescence (CL) detection. This method was based on enhance effect of FA on the CL reaction between luminol and BrO(-) in alkaline aqueous solution. Optimal separation and determination was obtained with an electrophoretic buffer of 35 mM sodium borate (pH 9.4) containing 0.8 mM luminol, and an oxidizer solution of 1.6 mM NaBrO in 100 mM NaCO(3) buffer solution (pH 12.0). Under the optimal conditions, the determination of FA was achieved in less than 20 min, and the detection limit was 2.0 x 10(-8) M (S/N=3). The relative standard deviations (RSDs) on peak area and migration time were in the 1.5 and 1.1%, respectively. The present CE-CL method was applied to the determination of FA in commercial pharmaceutical tablets, apple juices and human urine.     

Determination of domoic acid by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis--capillary zone electrophoresis (cITP-CZE) method for the determination of domoic acid in shellfish and algae is described. The optimised cITP-CZE electrolyte system was 10 mM HCl + 20 mM beta-alanine (BALA) + 0.05% hydroxyethylcellulose (leading electrolyte), 5 mM caproic acid (terminating electrolyte) and 20 mM caproic acid + 20 mM BALA + 0.1% HPMC (background electrolyte). A clear separation of the domoic acid from the other components of methanolic sample extract was achieved within 25 min. Method characteristics, i.e., linearity (0-200 microg/l), accuracy (recovery 101+/-3%), intra-assay repeatability (2.4%) and detection limit (1.5 microg/l) were determined. Speed of analysis, low laboriousness, high sensitivity and low running cost are the typical attributes of the cITP-CZE method. Developed method was successfully applied to analysis of shellfish samples and food supplements containing algae extract.     

Determination of carbohydrates by capillary electrophoresis with electrospray-mass spectrometric detection.

A method for the determination of underivatized carbohydrates using capillary electrophoresis (CE) with detection by electrospray ionization-mass spectrometry (ESI-MS) presented. Highly alkaline carrier electrolytes based on volatile organic bases like is diethylamine (DEA) combined with MS detection in the negativ-ion mode proved to be the optimum solution for the separation and detection of these analytes. Optimization of the carrier electrolyte composition has been performed with respect to its pH, ionic strength as well as the addition of an organic modifier. The influence of the DEA concentration in the sheath liquid on parameters like peak shapes or signal-to-noise (S/N) ratios was also investigated. Limits of detection (LOD) were in the range of 0.5-3.0 mgL(-1) and calibration was linear over an order of magnitude for almost all solutes investigated. Finally, the applicability of this method for the analysis of real samples was demonstrated with wine samples.     

Determination of egg white lysozyme by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis - capillary zone electrophoresis method for the determination of lysozyme in selected food products is described. The optimized electrolyte system consisted of 10 mM NH(4)OH + 20 mM acetic acid (leading electrolyte), 5 mM epsilon -aminocaproic acid +5 mM acetic acid (terminating electrolyte), and 20 mM epsilon -aminocaproic acid +5 mM acetic acid +0.1% m/v hydroxypropylmethylcellulose (background electrolyte). A clear separation of lysozyme from other components of acidic sample extract was achieved within 15 min. Method characteristics, i.e., linearity (0-50 micrograms/mL), accuracy (recovery 96+/-5%), intra-assay (3.8%), quantification limit (1 microgram/ml), and detection limit (0.25 microgram/mL) were determined. Low laboriousness, sufficient sensitivity and low running costs are important attributes of this method. The developed method is suitable for the quantification of the egg content in egg pasta.     

Determination of glycyrrhizin in liqueurs by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis-capillary zone electrophoresis method for the determination of glycyrrhizin in liqueurs is described. The optimised electrolyte system was 5 mM HCl+11 mM varepsilon-aminocaproic acid+0.05% hydroxyethylcellulose+30% methanol (leading electrolyte), 5 mM caproic acid+30% methanol (terminating electrolyte) and 20 mM caproic acid+10 mM histidine+0.1% hydroxyethylcellulose+30% methanol (background electrolyte). Method characteristics, i.e., linearity (20-500 ng/ml), accuracy (recovery 99+/-4%), intra-assay repeatability (2%), intermediate repeatability (3.8%) and detection limit (8 ng/ml) were determined. Speed of analysis, low laboriousness, high sensitivity and low-running cost are the typical attributes of the capillary isotachophoresis-capillary zone electrophoresis method. Developed method was successfully applied to analysis of liqueurs with liquorice extract and some foods (sweets and food supplements) containing liquorice. Found levels of glycyrrhizin in liqueurs, sweets and food supplements varied between 1-16 mg/l, 850-1050 mg/kg and 1.6-1.8 g/kg, respectively.     

Indirect UV detection of carbohydrates in capillary zone electrophoresis

Summary A new system for the rapid and sensitive analysis of underivatized carbohydrates has been established using capillary zone electrophoresis with indirect UV detection. At an applied potential of 28 kV, sugars and sugar acids could be separated by the combined effects of electroendosmosis and electrophoresis within 20 minutes in a fused silica capillary of 50 m internal diameter and an effective length of 100 cm using 6mM sorbic acid, pH 12.1, as both carrier electrolytie and chromophore. The alkaline pH ensured ionization of the sugars and, hence, their detection by means of charge displacement. Furthermore, the chosen concentration of sorbic acid allowed the smallest fractional change in the background signal to be measured. While the electrophoretic mobilities of the sugars were found to increase within a pH range of 11.9 to 12.3, those of the sugar acids were not affected. Due to the increasing competition of hydroxide ions in the displacement of the chromophore with rising pH, a significant loss of sensitivity is observed at pH values higher than 12.1 and this pH was found to provide sufficient resolution, optimum sensitivity, and a acceptably short analysis time. Under these conditions, a lower detection limit of 2 pmol was obtained for glucose.     

Determination of peroxides by capillary zone electrophoresis with amperometric detection

The combination of cathodic amperometric detection with capillary zone electrophoresis is demonstrated to be a versatile method for the quantification of organic and inorganic peroxides. A gold microelectrode, polarized at -600 mV against an Ag/AgCl reference electrode, is placed at the end of the capillary. Since the electroosmotic flow purges the detector electrode from oxygen, no degassing of the detector cell or the sample is necessary. With an injection volume of ca. 1 nl, hydrogen peroxide, peroxosulfate, peroxy alkanoic acids and the hydroperoxides of linoleic acid can be detected down to 10 micromol/l. Separation of the isomeric hydroperoxides of the unsaturated fatty acids is achieved by addition of beta-cyclodextrin to the electrolyte.     

Determination of quinolone antibiotics in urine by capillary electrophoresis with chemiluminescence detection

A novel and simple method is presented for the determination of norfloxacin, ciprofloxacin, and ofloxacin by capillary electrophoresis with chemiluminescence detection. This method is based on the enhancing effect of quinolones on the chemiluminescence reaction of the Ce(SO₄)₂–Ru(bpy)₃²⁺–HNO₃ system. Three quinolones were successfully separated and detected under optimum conditions. The obtained detection limits were 2.3×10^–7 mol/L, 5.2×10^–8 mol/L, and 7.8×10^–8 mol/L for ciprofloxacin, norfloxacin, and ofloxacin, respectively. The RSD of migration time and peak area were less than 1.8 and 3.8% (n = 5), respectively. The applicability of the proposed method was illustrated in the determination of ofloxacin in eye drops and of norfloxacin in human urine samples, and the monitoring of pharmacokinetics for norfloxacin.     

Determination of fumaric acid in apple juice by on-line coupled capillary isotachophoresis-capillary zone electrophoresis with UV detection

An on-line coupled capillary isotachophoresis-capillary zone electrophoresis (cITP-CZE) method for the determination of the fumaric acid content in apple juice is presented. A clear separation of fumaric acid in real samples is achieved within 20 min. The leading, terminating and background electrolyte of the employed system consist of 10 mM HCl+beta-alanine+5 mM beta-cyclodextrin+0.05% hydroxypropylmethylcelullose (HPMC), pH 3, 10 mM citric acid and 20 mM citric acid+beta-alanine+5 mM beta-cyclodextrin+0.1% HPMC, pH 3.3, respectively. The linearity, recovery, repeatability and detection limit of the developed method are 25-1000 ng/ml, 1.07%, 95.4+/-3.5 (+/-s)% and 10 ng/ml, respectively. Low laboriousness (no sample pretreatment), sufficient sensitivity and low running cost are the important attributes of the cITP-CZE method which was successfully applied to analyses of real samples of apple juices.     

Determination of diclofenac sodium by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the determination of diclofenac sodium using an end-column amperometric detection with a carbon fiber microelectrode, at a constant potential of 0.83 V vs. saturated calomel electrode. The optimum conditions of separation and detection are 4.90 x 10(-3) mol/l Na2HPO4-3.10 x 10(-3) mol/l NaH2PO4 (pH 7.0) for the buffer solution, 10 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, respectively. The limit of detection is 2.5 x 10(-6) mol/l or 5.2 fmol (S/N=2). The relative standard deviation is 0.8% for the migration time and 4.7% for the electrophoretic peak current. The method was applied to the determination of diclofenac sodium in human urine.     

Determination of biogenic amines by capillary zone electrophoresis with conductometric detection

A capillary zone electrophoresis (CZE) method with conductometric detection of biogenic amines (cadaverine, putrescine, agmatine, histamine, tryptamine and tyramine) is described. The optimised background electrolyte was the following: 15 mM histidine + 5 mM adipic acid + 1.5 mM sulphuric acid + 0.1 mM ethylenediaminotetraacetic acid + 0.1% hydroxyethylcellulose + 50% methanol. A clear separation of six biogenic amines from other components of acidic sample extract was achieved within 10 min. Method characteristics, i.e., linearity (0-100 micromol/ml), accuracy (recovery 86-107%), intra-assay repeatability (2-4%), and detection limit (2-5 micromol/l) were evaluated. Low laboriousness, sufficient sensitivity, speed of analysis, and low running cost are important attributes of this method. The developed method was successfully applied on the determination of biogenic amines in selected food samples.     

Determination of histamine by capillary zone electrophoresis with amperometric detection.

Capillary zone electrophoresis was employed for the determination of histamine using end-column amperometric detection with a carbon fiber microelectrode, at a constant potential. The optimum conditions of separation and detection were 10 mmol/L phosphate buffer, pH 5.6 for the buffer solution, 15 kV for the separation voltage, and 1.35 V (versus SCE) for the detection potential. The linear range was from 6.3 x 10(-7) to 1.5 x 1(-5) mol/L with the regression coefficient of 0.9997, and the detection limit was 4.0 x 10(-7) mol/L (S/N = 3). The proposed method was successfully applied to the direct determination of histamine in the beer samples without any sample clean-up procedures.     

Detection of paracetamol by capillary electrophoresis with chemiluminescence detection

Indirect detection of paracetamol was accomplished using a capillary electrophoresis-chemiluminescence (CE-CL) detection system, which was based on its inhibitory effect on a luminol-potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) CL reaction. Paracetamol migrated in the separation capillary, where it mixed with luminol included in the running buffer. The separation capillary outlet was inserted into the reaction capillary to reach the detection window. A four-way plexiglass joint held the separation capillary and the reaction capillary in place. K₃[Fe(CN)₆] solution was siphoned into a tee and flowed down to the detection window. CL was observed at the tip of the separation capillary outlet. The CL reaction of K₃[Fe(CN)₆] oxidized luminol was employed to provide the high and constant background. Since paracetamol inhibits the CL reaction, an inverted paracetamol peak can be detected, and the degree of CL suppression is proportional to the paracetamol concentration. Maximum CL signal was observed with an electrophoretic buffer of 30 mM sodium borate (pH 9.4) containing 0.5 mM luminol and an oxidizer solution of 0.8 mM K₃[Fe(CN)₆] in 100 mM NaOH solution. Under the optimal conditions, a linear range from 6.6 × 10⁻¹⁰ to 6.6 × 10⁻⁸ M (r = 0.9999), and a detection limit of 5.6 × 10⁻¹⁰ M (signal-to-noise ratio = 3) for paracetamol were achieved. The relative standard deviation (R.S.D.) of the peak area for 5.0 × 10⁻⁹ M of paracetamol (n = 11) was 2.9%. The applicability of the method for the analysis of pharmaceutical and biological samples was examined.     

Determination of cyclodextrins and their derivatives by capillary electrophoresis with indirect UV and conductivity detection

A fast and simple capillary electrophoretic method suitable for the determination of native alpha-, beta-, gamma-cyclodextrins, their randomly substituted tert-butyl derivatives (average degree of substitution 3.8-4.4), heptakis (2,6-di-O-methyl)- and heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin was developed. Naphthyl-2-sulfonic acid (2-NSA), 3-iodobenzoic acid (3-IBA) and (1S)-1-phenylethylamine (PHEA) were tested as selective complex forming and UV absorbing background electrolyte additives. The composition of optimized background electrolyte for the separation of uncharged cyclodextrins and their derivatives was: 15 mM 3-iodobenzoic acid titrated with tris[hydroxymethyl]aminomethane to pH 8.0, 5% (v/v) of acetonitrile. A complete resolution of mono-2-O-, mono-3-O- and mono-6-O-carboxymethyl-beta-cyclodextrin regioisomers was achieved in the optimized background electrolyte system: 40 mM PHEA titrated with 2-[N-morpholino]ethanesulfonic acid to pH 5.6. In addition to indirect UV detection a contactless conductometric detector was successfully utilized.     

Determination of cyclodextrins and their derivatives by capillary electrophoresis with indirect UV and conductivity detection

A fast and simple capillary electrophoretic method suitable for the determination of native α-, β-, γ-cyclodextrins, their randomly substituted tert-butyl derivatives (average degree of substitution 3.8 – 4.4), heptakis (2,6-di-O-methyl)- and heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin was developed. Naphthyl-2-sulfonic acid (2-NSA), 3-iodobenzoic acid (3-IBA) and (1S)-1-phenylethylamine (PHEA) were tested as selective complex forming and UV absorbing background electrolyte additives. The composition of optimized background electrolyte for the separation of uncharged cyclodextrins and their derivatives was: 15 mM 3-iodobenzoic acid titrated with tris[hydroxymethyl]aminomethane to pH 8.0, 5% (v/v) of acetonitrile. A complete resolution of mono-2-O-, mono-3-O- and mono-6-O-carboxymethyl-β-cyclodextrin regioisomers was achieved in the optimized background electrolyte system: 40 mM PHEA titrated with 2-[N-morpholino]ethanesulfonic acid to pH 5.6. In addition to indirect UV detection a contactless conductometric detector was successfully utilized.     

Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

In the present work, chemiluminescence detection was integrated with capillary electrophoresis microchip. The microchip was designed on the principle of flow-injection chemiluminescence system and capillary electrophoresis. It has three main channels, five reservoirs and a detection cell. As model samples, dopamine and catechol were separated and detected using a permanganate chemiluminescent system on the prepared microchip. The samples were electrokinetically injected into the double-T cross section, separated in the separation channel, and then oxidized by chemiluminescent reagent delivered by a home-made micropump to produce light in the detection cell. The electroosmotic flow could be smoothly coupled with the micropump flow. The detection limits for dopamine and catechol were 20.0 and 10.0 μM, respectively. Successful separation and detection of dopamine and catechol demonstrated the distinct advantages of integration of chemiluminescent detection on a microchip for rapid and sensitive analysis.