The measurement of average hydrodynamic velocity in capillary zone electrophoresis

Summary Based on the linear relationship between large sample injection time during hydrodynamic injection and migration time in non-stacking runs, a new method is proposed to measure average hydrodynamic velocity in capillary zone electrophoresis (CZE). Only the migration times for large injections need to be measured in this new approach, so the method is simple. Additionally, a modified Poiseuille equation is proposed to eliminate the deviation involved in Poiseuille’s equation. The average hydrodynamic velocity values determined by our method generally compare favorably with those determined by the modified Poiseuille equation.     

Capillary electrophoresis of synthetic peptide standards varying in charge and hydrophobicity

A mixture of eight structurally closely related synthetic peptides as capillary electrophoretic (CE) standards is introduced. The almost identical mass-to-charge ratio of the standards, coupled with their random-coil (i.e., no secondary structure) nature, offer a potent analytical test for CE to separate peptides varying only subtly in hydrophobicity. Parameters varied to effect a separation included background electrolyte concentration, temperature, applied voltage in capillary zone electrophoresis (CZE in uncoated capillaries), as well as the introduction of hydrophobic mechanisms to the separation either through the use of micelles or C8-coated capillaries. Our step-by-step approach culminated in an optimized combination of a CZE mechanism for separation of differently charged peptide groups (based on common mass-to-charge ratio) and an ion-pairing mechanism (effecting a separation within each group of identically charged peptides), which we have termed ion-interaction CZE or II-CZE. The study clearly shows how the peptide standards allow an excellent assessment of the resolving power of CE.     

Reduction in sample injection bias using pressure gradients generated on chip

Sample injection in microchip-based capillary zone electrophoresis (CZE) frequently rely on the use of electric fields which can introduce differences in the injected volume for the various analytes depending on their electrophoretic mobilities and molecular diffusivities. While such injection biases may be minimized by employing hydrodynamic flows during the injection process, this approach typically requires excellent dynamic control over the pressure gradients applied within a microfluidic network. The current article describes a microchip device that offers this needed control by generating pressure gradients on-chip via electrokinetic means to minimize the dead volume in the system. In order to realize the desired pressure-generation capability, an electric field was applied across two channel segments of different depths to produce a mismatch in the electroosmotic flow rate at their junction. The resulting pressure-driven flow was then utilized to introduce sample zones into a CZE channel with minimal injection bias. The reported injection strategy allowed the introduction of narrow sample plugs with spatial standard deviations down to about 45 μm. This injection technique was later integrated to a capillary zone electrophoresis process for analyzing amino acid samples yielding separation resolutions of about 4–6 for the analyte peaks in a 3 cm long analysis channel.     

Application of capillary electrophoresis in the quality control of osmotically treated water

Summary This paper discusses the use of capillary zone electrophoresis (CZE) with indirect UV detection for the separation and detection of ions. By use of a 70 cm×75 μm i.d. capillary at −15 kV and an electrophoretic buffer containing sodium chromate, 1-butanol, and cetyltrimethylammonium bromide as electroosmotic-flow modifier (pH 8) nine inorganic and organic anions were separated in less than 10 min. By use of the same type of capillary at 20 kV and an electrophoretic buffer containing imidazole and 18-crown-6 (pH 5) eight cations were separated in less than 5 min. The different variables that affect the separation were studied and optimized; the compounds were detected at low mg L⁻¹ levels after hydrodynamic injection under pressure. The method was tested with osmotically treated waters, and the results compared with those obtained by ion chromatography for anion analysis and by atomic absorption spectroscopy for cation analysis.     

DNA fragment separations by on‐line combination of capillary isotachophoresis–capillary zone electrophoresis with UV detection

A high‐speed DNA fragment separation system based on an on‐line combination of capillary ITP with CZE (CITP‐CZE) and using UV detection at 260 nm was developed. A novel CITP‐CZE buffer system of pH 6.1 was designed for the separation of ten DNA fragments with sizes ranging from 100 to 1000 bp. An effect of underivatized α‐, β‐ and γ‐cyclodextrins on the resolution of DNA fragments in the CZE step of the CITP‐CZE combination was systematically investigated. Methylhydroxyethylcellulose present in the BGE was used to eliminate the EOF. DNA ladder fragments were separated within 10 min with LODs in the range of 1–5 ng/μL (S/N = 3). The RSDs of the migration time and peak area of individual DNA fragments were in the range of 1–3 and 3–9%, respectively. The developed CITP‐CZE system was further applied to the analysis of digest plasmid DNA samples.     

Validation of a capillary zone electrophoresis method for the comparative determination of etoricoxib in pharmaceutical formulations

A CZE method was developed and validated for the analysis of etoricoxib in pharmaceutical dosage forms, using prilocaine as an internal standard. The CZE method was carried out on a fused-silica capillary (50 microm id, effective length 40 cm). The BGE consisted of 25 mM tris-phosphate solution at pH 2.5. The capillary temperature was maintained at 35 degrees C, the applied voltage was 25 kV, the injection was performed using the pressure mode at 50 mbar for 5 s, with detection at 234 nm using a photodiode array detector. The method was linear in the range of 2-150 microg/mL (r(2) = 0.9999). The specificity and stability-indicating capability were proven through the degradation studies and showing also that there was no interference of the excipients of the formulation. The accuracy was 99.49% with RSD of 0.66%. The limits of quantitation and detection were 2 and 0.58 microg/mL, respectively. Moreover, method validation demonstrated acceptable results for the precision, sensitivity, and robustness. The proposed method was successfully applied for the quantitative analysis of etoricoxib pharmaceutical formulations, and the results compared to the HPLC and LC-MS/MS methods, showing nonsignificant difference (p >0.05).     

Fast separation and determination of carnosic acid and rosmarinic acid in different rosemary (Rosmarinus officinalis) extracts by capillary zone electrophoresis with ultra violet-diode array detection

Summary Carnosol, carnosic acid, rosmarinic acid and other not identified phenolic compounds were separated by capillary zone electrophoresis (CZE) using a 40-cm long capillary and a 20 mM tetraborate buffer (pH 9.0), within 3 min. A UV-diode array detector was employed to collect spectra of phenolic compounds. The effect of some separation parameters on peak resolution and migration time of phenolic species present in a refined rosemary extract was studied. The repeatability of the method was also investigated: the intraday relative standard deviation on total peak area was less than 4%, while the intraday relative standard deviation on migration time was less than 0.6%. Moreover the CZE method showed good sensitivity (0.0007 μg mL¹ for carnosic acid and rosmarinic acid). Carnosic acid and rosmarinic acid have been quantified in different commercial extracts of rosemary. Finally, the optimized method was also applied to evaluate the recovery of these two compounds when different organic solvents were employed during the extraction procedure.     

Study of the Electrophoretic Behaviour of Flavonoids

The electrophoretic behaviour of 13 flavonoids commonly found in medicinal plants has been studied. The performance of two modes of separation, capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC), was assessed and compared. MEKC was found to be more effective for separation of the analytes. The effect of structure on the electrophoretic behaviour of the flavonoids is also discussed.     

Comparative study of capillary zone electrophoresis and micellar electrokinetic chromatography for the separation of twelve aromatic sulphonate compounds

Summary Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), were investigated for the separation of 12 aromatic sulphonate compounds. In CZE, although the voltage applied, the buffer concentration and the pH were optimized for effective separation of the compounds studied, under the best conditions four of the five amino compounds coeluted, as did naphthalene-1-sulphonic acid and naphthalene-2-sulphonic acid. In MEKC, sodium dodecyl sulphate (SDS) and Brij 35 were chosen as the anionic and nonionic surfactants and the effect of the concentration of micelles was examined. The effect of adding methanol as the organic modifier was also investigated with each of these micellar systems. All the analytes, including the isomers, were completely separated by use of MEKC with Brij 35 but when SDS was used only 11 compounds were separated because two amino compounds coeluted.     

胶束在线扫集毛细管电泳法测定三聚氰胺

研究胶束在线扫集毛细管电泳法测定三聚氰胺的可行性,结果表明,与区带毛细管电泳相比,胶束在线扫集毛细管电泳法富集倍数提高约60倍。缓冲体系为140 mmol/L SDS+20 mmol/L NaH2PO4(pH 2.20)+10%(体积分数)甲醇,分离电压-18 kV,进样时间30 s,测量波长214 nm。考察了SDS浓度、pH、进样时间、运行电压等因素对分离测定的影响情况。在优化条件下,三聚氰胺在9 min时出峰,峰面积RSD≤3.7%。方法检出限、线性范围、相关系数分别为:0.13μg/mL、0.50~32.0μg/mL、0.9997。方法可用于奶粉中三聚氰胺的分离测定。     

A sulfonated capillary that gives reproducible migration times for capillary zone electrophoresis and micellar electrokinetic chromatography

To obtain reproducible migration times and rapid analyses of analytes, sulfonate groups were chemically introduced to the inner wall of untreated fused-silica capillary with 2-(4-chlorosulfonylphenyl)ethyltrichlorosilane. The sulfonated capillary showed relatively constant electroosmotic mobility which was greater than that obtained by an untreated fused-silica capillary over the pH range studied (pH 2-9). In both CZE and MEKC, the RSDs of the migration times of analytes with the sulfonated capillary were less than 0.2% which were significantly lower than those obtained with an untreated fused-silica capillary (0.5-3.5%). When BGE were set at pH 7.0 for CZE and MEKC, the analysis times with the sulfonated capillary were about half those obtained with an untreated fused-silica capillary. These results indicate that the sulfonated capillary can provide highly reproducible and rapid analyses in CE.     

Investigation of selected parameters of capillary zone electrophoresis method for analysis of isolates of outer membrane vesicles

The capillary zone electrophoresis (CZE) has recently been proposed by our group as a novel technique for outer membrane vesicles (OMVs) characterization (J. Chromatography 1621 (2020) 461047). In present work the impact of selected parameters of CZE method on OMVs isolates analysis was assessed. It was shown that the extension of sample injection plug length significantly improves the detectability of macromolecular aggregates in CZE. Moreover, a negligible adsorption of OMVs to both uncoated and polymer-modified (poly(DMA-GMA-MAPS)) capillary walls was proven. Finally, the relaxation effect as well as deformation/polarization of vesicles were demonstrated to affect OMVs electrophoretic mobility. The significance of these findings was discussed.     

Capillary electrophoresis of DNA for molecular diagnostics

A number of applications of capillary zone electrophoresis (CZE) in sieving liquid polymers (notably linear polyacrylamides and cellulose) for the analysis of polymerase chain reaction (PCR) products of clinically relevant, diagnostic DNA, are reviewed. The fields covered are: human genetics, quantitative gene dosage, microbiology and virology, forensic medicine and therapeutic DNA (notably, antisense nucleotides). Some unique, novel developments are highlighted, such as: (i) nonisocratic CZE, i.e., temperature-programmed CZE for detection of DNA point mutations; (ii) the synthesis of novel N-substituted acrylamides, offering extreme resistance to alkaline hydrolysis coupled to high hydrophilicity. In the field of denaturing gradient gel electrophoresis (DGGE), as routinely performed in gel slabs, a novel methodology is described in CZE: double-gradient DGGE. In this technique, two gradients are simultaneously applied along the migration direction: a chemical (or thermal) denaturing gradient, for partially unwinding homo- and hetero-duplexes of DNA, and a porosity gradient, for recompacting diffuse bands melting over a broader range of denaturing conditions. It is thus demonstrated that chemical gradients, in addition to temperature gradients, can be easily implemented even in a capillary format.     

场放大样品进样-压力辅助毛细管区带电泳测定饮用水中的百草枯

1 引 言 百草枯属有机杂环类季铵盐除草剂,由于它具有优良的除草效果,已广泛应用于多种作物的杂草防治.百草枯具有极强的水溶性,极易迁移至水体环境中,从而对饮用水的质量安全构成潜在威胁.目前,百草枯的残留检测方法主要有分光光度法[1]、液相色谱-质谱联用法[2]、气相色谱质谱联用法[3]和毛细管电泳法(CE) [4～6].采用分光光度法测定百草枯,不仅操作繁琐费时,而且灵敏度低.采用气相色谱法测定百草枯,通常需要衍生化,应用较少[3].采用液相色谱法测定百草枯,通常需要在流动相中添加离子对试剂[2].毛细管电泳具有分离效率高,分析速度快等优点,已被广泛用于水样中百草枯残留的测定.然而,毛细管电泳灵敏度不高,极大地限制了其在实际样品分析中的应用.场放大样品进样(FASI)是一种简单有效的在线富集方法,其富集倍数可达1000倍[7],可有效提高毛细管电泳技术的灵敏度,因此应用较为广泛.本实验建立了场放大样品进样-压力辅助毛细管区带电泳法(CZE),用于测定饮用水中百草枯的残留量.     

Capillary electrophoresis for the direct determination of cephalosporins in clinical samples

Summary The possibility of determination of four cephalosporin antibiotics in clinical samples by capillary electrophoresis has been investigated. The separation conditions for capillary zone electrophoresis (CZE) were studied in detail. The precision of migration times measured by use of the optimized method was satisfactory (RSD<1%) and response was linearly dependent on concentration over the approximate range 2–150 mg L⁻¹ for all the compounds studied (cefuroxime, cefotaxime, ceftriaxone, and ceftazidime). Complete separation could be achieved within 5 min. The CZE method was found to be highly suitable for direct determination of the antibiotics in clinical samples such as wound drainage, cerebrospinal fluid, and urine; for serum, however, the use of micellar electrokinetic capillary chromatography (MECC) was more advantageous. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Comparison of field‐enhanced and pressure‐assisted field‐enhanced sample injection techniques for the analysis of water‐soluble vitamins using CZE

A new online concentration method, namely pressure‐assisted field‐enhanced sample injection (PA‐FESI), was developed and compared with FESI for the analysis of water‐soluble vitamins by CZE with UV detection. In PA‐FESI, negative voltage and positive pressure were simultaneously applied to initialize PA‐FESI. PA‐FESI uses the hydrodynamic flow generated by the positive pressure to counterbalance the reverse EOF in the capillary column during electrokinetic sample injection, which allowed a longer injection time than usual FESI mode without compromising the separation efficiency. Using the PA‐FESI method, the LODs of the vitamins were at ng/mL level based on the S/N of 3 and the RSDs of migration time and peak area for each vitamin (1 μg/mL) were less than 5.1%. The developed method was applied to the analysis of water‐soluble vitamins in corns.     

Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase

Cellobiohydrolase (CBH) is an important enzyme for the conversion of lignocellulosic biomass to ethanol. This work separated the glycoforms of CBH possessing different numbers of neutral mannoses using capillary zone electrophoresis (CZE) in a 50 mM, pH 7.5 phosphate buffer. The method analysed CBH in an intact form using a polyacrylamide coated fused silica capillary without requiring additives or labelling of the enzyme. The migration time of the major peak was found to be 21.6±0.1 min (n=3) and the approach is suitable for testing of batch-to-batch consistency of CBH. Ease-of-use, automation and speed are the other benefits due to which the use of CZE for analysing glycoforms of CBH was concluded to be ideal.     

Development of a capillary electrophoresis system coupled to sequential injection analysis and evaluation by the analysis of nitrophenols

A combination of a laboratory-made capillary electrophoresis system and a sequential injection analysis equipment is described. For characterization, the system was successfully applied to the separation and quantification of nitrophenols. A blue LED was used as light source, and hydrodynamic injection was carried out by using a pressure-stable solenoid valve and an inflatable pressure reservoir. A good reproducibility of migration time (0.5%) and peak heights (5%) were obtained. The calibration by using peak heights was found to be linear up to 776?µmol?L^?1 for all three compounds. The system was robust and reliable for autonomous analysis without observation. All maintenance requirements including the conditioning of the capillary and flushing of both buffer reservoirs were carried out automatically. Instrumentation aspects of the capillary electrophoresis part are compared with former described hyphenated flow systems showing maximal operation versatility. Instrumental control and data evaluation were carried out using the software package AutoAnalysis.     

Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Amylose tris(5-chloro-2-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSP) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the nature of silica, the particle size and pore diameter, the chiral selector loading onto silica, the mobile phase composition and pH, as well as separation variables such as a linear flow rate of the mobile phase, applied voltage in CEC, etc. on the separation of enantiomers was studied. It was found that CSPs based on amylose tris(5-chloro-2-methylphenylcarbamate) can be used for preparation of stable capillary columns for enantioseparations by nano-LC and CEC in combination with polar organic and aqueous–organic mobile phases. Higher peak efficiency was observed in CEC than in nano-LC.     

毛细管电泳法和高效液相色谱法拆分联萘二酚酸对映体

采用毛细管电泳法和高效液相色谱法直接拆分2,2′-二羟基-1,1′-联二萘-3,3′-二甲酸(HBNC)对映体.以四种不同的β-环糊精为手性添加剂,考察环糊精的种类与浓度、缓冲液pH值及浓度、分离电压、温度等因素对HBNC分离的影响.结果表明:采用10 mmol/L磺丁基醚-β-环糊精+20 mmol/L磷酸盐缓冲液(pH=7.0),20 kV分离电压,HBNC对映体在20 min内达到基线分离,分离度达到3.31.采用(S)-叔-亮氨酸基-(S)-1-(α-萘基)乙胺手性柱,正己烷-乙醇-三氟乙酸(97∶3∶0.2,V/V)流动相,HBNC对映体在40 min内也基本达到基线分离.