Study of an electroosmotic pump for liquid delivery and its application in capillary column liquid chromatography

A packed-bed electroosmotic pump (EOP) was constructed and evaluated. The EOP consisted of three capillary columns packed in parallel, a gas-releasing device, Pt electrodes and a high-voltage power supply. The EOP could generate output pressure above 5.0 MPa and constant flow rate in the range of nl/min to a few microl/min for pure water, pure methanol, 2 mM potassium dihydrogenphosphate buffer, the buffer-methanol mixture and the pure water-methanol mixture at applied potentials less than 20 kV. The composition of solvent before/after pumping was quantitatively determined by using a gas chromatograph equipped with both flame ionization detector and thermal conductivity detector. It was found that there were no apparent changes in composition and relative concentrations after pumping process for a methanol-ethanol-acetonitrile mixture and a methanol-water mixture. Theoretical aspect of the EOP was discussed in detail. An capillary HPLC system consisting of the EOP, an injection valve, a 15 cm x 320 microm i.d., 5 microm Spherigel C18 stainless steel analytical column, and an on-column UV detector was connected to evaluate the performance of the EOP. A comparative study was also carried out with a mechanical capillary HPLC pump on the same system. The results demonstrated that the reproducibility of flow rate and the pulsation-free flow property of the EOP are superior to that of mechanical pump in capillary HPLC application.     

Comparison of chromium speciation by CZE and ion exchange followed by AAS

The hydrogen chromate anion (HCrO4-), which is the predominant species in acidic solutions and solutions with low chromium concentration, was determined by capillary zone electrophoresis (CZE) using UV detection on-column at 200 nm. A fused-silica capillary (55 cm x 50 microm i.d.) was employed with a high negative voltage of 20 kV. Total chromium was determined after reduction by H2O2 and its complexation by EDTA. The use of H2O2 as reducing agent is advantageous, as it does not increase the conductivity of the solution. Detection limits achieved (for 200 s injection time) were 30 and 8 microg/L for Cr(VI) and Cr(III), respectively. The CZE results obtained for Cr(III) and Cr(VI) were compared with those obtained by ion exchange with subsequent AAS.     

Capillary electrophoresis coupled with electrochemical detection using porous etched joint for determination of antioxidants

Capillary electrophoresis coupled with electrochemical detection (CE-EC) for determination of antioxidants, propyl gallate (PG) and tert-butylhydroquinone (TBHQ), in cosmetic samples was proposed in this work. A porous etched joint was used to isolate the electrochemical detection from the electrophoretic high voltage. Compared with the 25 microm i.d. capillary without a decoupler in a CE-EC system, a 75 microm i.d. capillary applied in the present system gave an improvement in both sample injection and sensitivity. Moreover, the carbon fiber working electrode could be directly in touch with the end of separation capillary due to the elimination of the effect of separation voltage on the EC detection, so the alignment of working electrode and capillary became easy and the dead volume was also decreased. Baseline separation of the two antioxidants was achieved by CE in a 50 cm long x 75 microm i.d. capillary at 20 kV using 5.0 mmol L(-1) phosphate buffer (pH 8.00). 0.7 V (versus Ag/AgCl) was applied to the carbon fiber electrode for electrochemical detection. Under the optimal condition, the precisions (RSD, n=4) of peak height and migration time of PG and TBHQ were 2.39-3.59% and 0.34-0.44%, respectively. The detection limits of PG and TBHQ were 2.51x10(-6) and 4.88 x 10(-6) mol L(-1) for standard solution and 0.0751 and 0.0328 mg g(-1) for the real cosmetic samples with consumption of 0.3g sample. Analysis of TBHQ and PG in cosmetics samples was also achieved with the present system and the spiked recoveries of two analytes in cosmetics samples were in the range of 93.6-98.8%.     

Analysis of thiocyanate in biological fluids by capillary zone electrophoresis

A new sensitive and simple method has been developed for the determination of thiocyanate in human serum, urine and saliva. The determinations were performed in a fused-silica capillary [64.5 cm (56 cm effective length) x 75 microm] using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity), temperature of capillary 25 degrees C and direct detection at 200 nm. Serum samples were 10-times diluted with deionised water and deproteinised with acetonitrile in the ratio 1:2. Urine and saliva samples need only 20-fold dilution with deionised water. The proposed method was successfully applied to the determination of thiocyanate in various human serum, saliva and urine samples.     

Determination of scopolamine, atropine and anisodamine in Flos daturae by capillary electrophoresis.

A capillary electrophoresis method was developed for the separation and determination of tropane alkaloids in Flos daturae plants. Separation was performed on a fused silica capillary(42.1 cm x 50 microm i.d.) at an applied voltage of 20 kV. Scopolamine, atropine and anisodamine were well separated in the buffer of 50 mmol/L phosphate buffer (pH 5.0) containing 20% (v/v) tetrahydrofuran (THF). Beer's law was obeyed in the range of concentration of 2.4-21.8 microg/mL for scopolamine, 4.0-36.0 microg/mL for atropine and 2.6-23.7 microg/mL for anisodamine, respectively, and the correlation coefficients were over 0.999 (n = 6). The developed method was applied for the analysis of herb samples.     

Robust monolithic silica-based on-chip electro-osmotic micro-pump

A robust, compact, on-chip, electro-osmotic micro-pump (EOP) for micro-flow analysis, based on parallel, encased, 10 x 0.1 mm I.D. monolithic silica capillary columns has been developed. A 15 x 40 x 2 mm poly(methyl methacrylate) (PMMA) chip, containing a total of nine parallel EOP systems was fabricated, allowing the use of single, double or triple monolithic columns to produce increased flow as required. The monolithic silica was compatible with both aqueous and organic solvents without swelling or shrinking problems, with the triple column EOP capable of generating flow of up to 0.6 microL min(-1) under zero pressure load and over 0.1 microL min(-1) with an applied pressure of ca. 2.4 bar using an applied voltage of just 2 kV. Current generated at the 2 kV applied voltage for a 2 mM acetate buffer solution (pH 4.5) was under 4 microA, allowing stable, bubble-free flow. The developed triple column EOP was incorporated within a micro-fluidic chip (5.0 x 2.0 x 0.4 cm) integrated with a second single 10 x 0.1 mm column EOP, for combined sample injection and simple on-chip micro-flow analysis.     

托吡卡胺对映体的毛细管电泳-方波安培分离检测

采用毛细管电泳-方波安培检测法,在熔融石英毛细管(75 μm i.d.×50 cm)中,以7 mmol/L 三羟甲基氨基甲烷(Tris)-10 mmol/L柠檬酸-2 mmol/L硼酸-15mmol/L β-环糊精 （β-CD） (pH 3.0)为电泳介质,采用重力进样,高度差为20 cm,进样时间为10 s,在分离电压为15 kV,方波平衡电位+0.8 V的条件下,实现了托吡卡胺对映体的分离检测。线性范围为5~750 μmol/L,检出限为2 μmol/L。对影响分离度的因素β-CD浓度、硼酸浓度及p     

Determination of quinolizidine alkaloids in Sophora flavescens and its preparation using capillary electrophoresis

A simple and accurate capillary electrophoresis method was developed for the determination of four quinolizidine alkaloids in Sophora flavescens and Kuhuang injection. Optimum separation of the analytes was obtained on a 65 cm x 75 microm i.d. uncoated fused-silica capillary using a aqueous buffer system of 60 mmol L(-1) sodium borate at pH 8.5, with applied voltage and capillary temperature of 12 kV and 25 degrees C, respectively. Detection wavelength was set at 204 nm and jatrorrhizine was used as the internal standard. Good linear relationships between peak-area ratios and concentrations of the analytes were observed over the concentration range 0.044-0.792 mg mL(-1) for matrine, 0.142-1.926 mg mL(-1) for oxymatrine, 0.0377-0.3393 mg mL(-1) for sophocarpine and 0.0664-1.062 mg mL(-1) for sophoridine. The recoveries of four alkaloids ranged between 93.08 and 101.4% with relative standard deviations from 0.7 to 9.2% (n = 6) as determined by standard addition. The limits of detection for four alkaloids were determined to be over the range 8.8-48.0 microg mL(-1). Contents of four alkaloids in Sophora flavescens and three alkaloids in Kuhuang injection were successfully determined under the optimum conditions.     

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.     

Determination of copper and iron using [S,S']-ethylenediaminedisuccinic acid as a chelating agent in wood pulp by capillary electrophoresis.

A capillary electrophoresis (CE) method is described for the simultaneous determination of copper and iron after complexation with a readily biodegradable chelating agent, [S,S']-ethylenediaminedisuccinic acid (EDDS), in wood pulp. CE separation was performed in a fused-silica capillary (50 microm i.d.; total length, 65 cm) with an electrolyte containing 25 mM borate buffer and 0.5 mM CTAB at pH 7.0 and an applied voltage of -25 kV. The samples were introduced by applying a 50 mbar pressure for 2 s, and detection of the complexes was monitored at 245 nm. The methodology performance of the methods was evaluated in terms of the linearity, limit of detection (LOD), limit of quantitation (LOQ) and reproducibility. The applicability of the method was demonstrated for the analysis of copper and iron in wood pulp.     

Development of a NACE method for simultaneous measurement of three adenosine monophosphate isomers in biomimicking prebiotic synthesis without sample pretreatment

A practical NACE method was developed for simultaneous determination of three adenosine monophosphate (AMP) isomers. Separation of three AMP isomers was achieved using 200 mM Tris/H(3)BO(3) in acetontrile/water (2:1 v/v) at pH* 10.0 as the running buffer and +25 kV as the applied voltage over a bare fused-silica capillary of 50 microm id x 375 microm od x 54.5 cm (46 cm to the detector window). At 260 nm, the calibration curves were linear in the range of 1-100 microg/mL. The detection limits were less than 0.70 microg/mL. The recovery ranged from 94.5 to 106.4%. The intraday RSDs of the migration times were between 2.1 and 3.0%. The developed NACE method has been successfully applied for the determination of three AMP isomers in the real samples of biomimicking prebiotic synthesis reaction between N-(O,O-diisopropyl) phosphoryl amino acid and adenosine.     

毛细管电泳法同时测定血清中的左旋多巴和甲基多巴

建立了毛细管电泳-紫外检测同时测定血清中左旋多巴和甲基多巴的方法。以40 mmol/L硼砂（pH 9.5）为分离缓 冲溶液,在3.45 kPa（0.5 psi）压力下进样7 s、分离电压22 kV、检测波长200 nm、温度25 ℃的条件下进行测定,两 种物质获得了较好的分离。甲基多巴和左旋多巴分别为1.0~64.0 mg/L和1.0~71.0 mg/L时与峰面积呈良好的线性关系, 线性相关系数分别为0.9998和0.9994,检出限分别为0.6和0.8 mg/L(以信噪比为3计)。将该法用于血清中甲基多巴和左 旋多巴的测定,回收率为82.8%~88.8%,相对标准偏差为2.10%~2.63%。     

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.     

Separation and determination of abused drugs clenbuterol and salbutamol from complex extractants in swine feed by capillary zone electrophoresis with simple pretreatment

A simple but efficient capillary zone electrophoresis (CZE) method was developed for the fast separation and determination of two misused beta2-agonists clenbuterol (CLB) and salbutamol (SAL) from complex background extractants existing in swine feed samples. The proper experimental conditions were achieved as 20.0 mmol/l pH 10.5 Na(2)HPO(4)-NaOH buffer, 20 kV applied voltage, fused-silica capillary of 60.5 cm x 75 microm i.d. (50 cm to detector). Under the proper conditions, the two abused drugs can be online isolated from the complex extractants and the separation between CLB and SAL is good, all of the target analytes can be detected within 4.5 min. The linear response of CLB and SAL concentration ranges from 2.0 to 100.0 microg/ml with high correlation coefficient (R(2)=0.9990) and (R(2)=0.9986), respectively. Limit of detection (LOD) and limit of quantification (LOQ) was calculated to be 0.95 and 3.17 microg/ml for CLB, 1.07 and 3.57 microg/ml for SAL. The precision values (expressed as R.S.D.) of intra- and inter-day were 1.24-2.36% and 0.90-3.85% for CLB, 0.47-1.64% and 0.91-3.46% for SAL. Recoveries spiked at levels 5.0, 25.0, 80.0 microg/ml ranged between 93.30% and 104.33% with R.S.D. less than 5%. Finally, the developed method has been applied to the analysis of real swine feed samples and has achieved satisfactory results.     

High-performance capillary zone electrophoretic assay for markers of diabetic nephropathy in plasma and urine

A new high-performance capillary zone electrophoretic assay for creatine (Cr), creatinine (Cn), urea (U) and uric acid (Ua), markers of human diabetic nephropathy, both in plasma and urine has been developed with UV detection at 200 nm. The plasma sample was deproteinized with trichloroacetic acid and centrifuged at 10 000 rpm for 10 min. The urine sample was diluted 20-fold with buffer before analysis. The optimum separation conditions for the markers was investigated with respect to the concentration of the buffer, the pH, the voltage and the capillary temperature. Baseline separation was achieved in 25 mmol/L phosphate buffer (pH 3.45) using a 21 cm x 75 microm I.D. fused-silica capillary at 40 degrees C with an electric field of 1190 V/cm. The calibration curves showed good linearity in the range 3.5-1000, 0.18-700, 500-5000 and 2-800 microM (r2 min > 0.998) for Cr, Cn, U and Ua, respectively. The proposed method also has a high reproducibility (peak area RSD max < 3%) and has been successfully applied to the determination of clinical samples.     

Rapid analysis of antimicrobial metabolites monoacetylphloroglucinol and 2,4-diacetylphloroglucinol using capillary zone electrophoresis

A rapid capillary electrophoretic (CE) method was developed for the determination of phloroglucinol compounds, monoacetylphloroglucinol (MAPG) and 2,4-diacetylphloroglucinol (DAPG), in microbial supernatants of Pseudomonas fluorescens F113 over a 24-h growth cycle. Prior to electrophoretic separation, solid-phase extraction of supernatant samples on octadecylsilica for the purpose of sample cleanup is recommended. The optimum electrophoretic conditions were found to be 25 mM sodium tetraborate running buffer at pH 9.3, temperature at 25 degrees C with an applied voltage of 25 kV. The capillary was an Agilent fused-silica capillary of total length 33 cm x 50 microm inner diameter, 375 microm outer diameter, with effective length 24.5 cm. While MAPG and DAPG were monitored at selected wavelengths in the range of 214-320 nm, analysis at 214 nm was used and a CE separation time of less than 2 min was achieved. A partial method validation study was performed in accordance with European Agency for Evaluation of Medicinal Products (EMEA) guidelines. The method displayed linearity over the investigated range of 10-200 microg/mL, with limits of detection of 1.2 microg/mL for MAPG and 1.3 microg/mL for DAPG.     

Development of a capillary electrophoresis method for the enantioselective estimation of primaquine in pharmaceutical formulations

A capillary electrophoresis (CE) method has been developed that allows the separation and estimation of primaquine enantiomers using hydroxypropyl-gamma-cyclodextrin (HP-gamma -CD) as a chiral selector. The influence of chemical and instrumental parameters on the separation, such as type and concentration of CD, buffer concentration, buffer pH, applied voltage, capillary temperature, and injection time, were investigated. Good separation of the racemic mixture of primaquine was achieved using a fused-silica capillary (52.5 cm effective length x 50 microm id) and a background electrolyte composed of tris-phosphate buffer solution (50 mM, pH 2.5) containing 15 mM HP-gamma-CD as a chiral selector. The recommended applied voltage, capillary temperature, and injection time were 15 kV, 25 degrees C, and 6 s, respectively. Within-day and interday reproducibility of peak area and migration time gave relative standard deviation values ranging from 1.05-3.30%. Good recoveries (range of 96.8-104.9%) were obtained from the determination of placebos that were spiked with 0.25-1.00 mg/L primaquine. The proposed CE method was successfully applied to the assay of primaquine diphosphate in pharmaceutical formulations (tablets).     

Simultaneous determination of delta-aminolevulinic acid, porphobilinogen, levulinic acid and glycine in culture broth by capillary electrophoresis.

Capillary electrophoretic simultaneous determination of a mixture containing delta-aminolevulinic acid, porphobilinogen, levulinic acid and glycine was investigated. With increases in the sodium tetraborate buffer concentration (5-70 mM), resolution of the four components was improved, but the migration time was increased. Alternatively, with increases in the applied voltage (5-22.5 kV), a shortened migration time was seen but this adversely affected resolution. The components were separated with high resolution by using a fused-silica capillary column (75 cm x 75 microm I.D.) filled with 30 mM sodium tetraborate buffer (pH 9.3-9.4) under the applied voltage of 20 kV (constant voltage mode). When the established method was applied to the culture broth of Rhodopseudomonas sphaeroides, a photosynthetic bacterium, the four components mentioned above were separated with good resolution. Furthermore, the use of this method would provide a fast, sensitive and specific method for monitoring the administration of delta-aminolevulinic acid in photodynamic cancer therapy, for the measurement of delta-aminolevulinic acid dehydratase activity in erythrocytes, and for testing the delta-aminolevulinic acid assay and for impurities in drug formulation.     

电渗泵在微柱液相上的应用

 设计评价了一种能够取代机械泵的电渗泵 (EOP) ,该泵可以产生 2 0MPa～ 6 0MPa的输出压力和几十nL/min～ 3μL/min的输出流量 ;用乙腈 水作为流动相 ,在 14cm× 32 0 μmi d (C18,5 μm )微柱上分离了萘、蒽、菲3种物质的混合试样 ,证明EOP在微柱液相领域有广阔的应用前景。     

Development and validation of a stability-indicating micellar electrokinetic chromatography method for the determination of ezetimibe in pharmaceutical formulations.

A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 microm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35 degrees C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2-150 microg/mL (R2=0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 microg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography method.