Separation of bacteria by capillary electrophoresis

Differences in the surface charges of bacteria can be exploited for their separation by capillary electrophoresis. Because of their low electrophoretic mobility, the separation is not always easy to perform, especially in the presence of the electroosmotic flow. Elimination of electroosmotic flow by capillary wall modification with γ‐(trimethoxysilyl)propyl methacrylate followed by acrylamide bonding permits separation over a distance of 8.5 cm.     

Protein separation by capillary gel electrophoresis: A review

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples.     

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.     

毛细管电泳安培法测定脂可平胶囊中的姜黄素

采用毛细管电泳柱端安培检测对脂可平胶囊中的姜黄素进行测定。着重研究了缓冲溶液浓度和酸碱度、检测电位、进样时间和高压对分离测定的影响。以微Pt电极为工作电极,电极电位为 1.0 V,以V(甲醇)∶V(乙醇)∶V(水)=5∶2∶3为非水介质,磷酸二氢钾和硼砂(pH 9.5)为缓冲体系,并用二阶样条小波进行滤波处理,姜黄素在1.0~120 mg/L范围内,峰高与其质量浓度呈良好的线性关系,线性回归方程:Y=20.2 146ρ,检出限为0.02 mg/L。     

高效毛细管电泳电导分离-检测亮氨酸对映体

以未涂层融硅石英毛细管(50 cm×75μm)为分离柱,2 mmol/L NaAc+2mmol/L HAc+0.5 mmol/L Cu2+(pH 4.0)作为电泳运行液,分离电压15 kV,建立了亮氨酸对映体的高效毛细管电泳-电导分离检测的方法。对缓冲溶液的种类、浓度、分离电压、有机改性剂等因素对分离的影响进行了讨论。L-亮氨酸和D-亮氨酸的线性回归方程分别为:y=5.998ρ+40.677,y=3.605ρ+42.087。线性范围:L-亮氨酸4.0~160 mg/L;D-亮氨酸6.0~160 mg/L。检出限分别为:1.5和2.0 mg/L。     

毛细管电泳安培法检测酚类化合物

使用自行设计组装的毛细管电泳柱端安培检测系统 ,对四个酚类化合物进行了分离检测。研究了工作电极、缓冲液及其 p H值、检测电压和分离电压对分离检测的影响。在优化条件下 ,4个酚在 5× 1 0 -6～ 5× 1 0 -4 mol/L范围内峰高与浓度成良好的线性关系 ,检测下限为 8.5× 1 0 -7mol/L     

The effect of aggregation on the separation performance of bacteria in capillary electrophoresis

During the last 15 years, methods for the capillary electrophoretic separation of different bacteria species have been developed, which exploit their characteristic cell surface‐charge to volume ratio. A special variant, the polymer‐based CE of bacteria, includes a focusing step, which forces the bacteria cells to form aggregates at the beginning of the electrophoretic process, resulting in very high apparent efficiencies. Our experiments presented in this article reveal that the migration time of bacteria species in polymer‐based CE increases with a growing amount of injected cells. Thus, the electrophoretic mobilities are not characteristic for the single cells of one species, but for the aggregates of the bacteria species, which are formed during the focusing process. Electrophoretic mobility (EM) data are obviously inapplicable for the identification of bacteria if the concentration of the bacteria sample solution is not constant. Fractions taken during the electrophoretic separation of different bacteria species were cultivated and tested for species purity. Interestingly, the electrophoretic bands were never pure, as all of them contained different mixtures of the injected species. We attribute this to the formation of stable mixed‐species aggregates during polymer‐based focusing. The mixed clusters migrate in the electric field with consistent velocity as a whole and are not separated electrophoretically.     

Profiling of erythropoietin products by capillary electrophoresis with native fluorescence detection

The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal‐to‐noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE‐Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO‐α and EPO‐β) based on the recorded glycoform pattern. The developed CE‐Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.     

Determination of dioxopromethazine hydrochloride by capillary electrophoresis with electrochemiluminescence detection

The paper presents a rapid method for the determination of dioxopromethazine hydrochloride (DPZ), an antihistamine drug, by the capillary electrophoresis with electrochemiluminescene detection (CE–ECL) using tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) reagent. This CE–ECL detection method has high sensitivity, good selectivity and reproducibility for DPZ analysis. Under the optimized conditions: separation capillary, 38 cm length (25 μm i.d.); sample injection, 10 s at 8 kV; separation voltage, 12.5 kV; running buffer, 20 mmol L⁻¹ sodium phosphate of pH 6.0; detection potential, 1.15 V; 50 mmol L⁻¹ of phosphate buffer (pH 7.14) containing 5 mmol L⁻¹ of Ru(bpy)₃²⁺ in ECL detection cell, the detection limit of DPZ was 0.05 μmol L⁻¹ (S/N = 3). The linear range extended from 5 to 100 μmol L⁻¹. The linear curve obtained was Y = 181.62 + 9.28X with a correlation coefficient of 0.9970. The relative standard deviations of the ECL intensity and the migration time for six continuous injections of 5 μmol L⁻¹ DPZ were 3.7% and 0.92%, respectively. The CE–ECL method was applied to analyze DPZ in real samples including tablets, rat serum and human urine, and satisfactory results were obtained without interference from samples matrix. The CE–ECL technique was proved to be a potential method for the detection of DPZ in clinic analysis.     

氯霉素的高效毛细管电泳分离-电导检测

在熔融石英毛细管(50μmi.d×50cm)中,以6mmol/LH3BO3 2mmol/L硼砂 体积分数0.02%TEPA(四乙烯五胺)为电泳运行介质,分离电压-15.0kV,采用毛细管电泳-电导检测法在4min内实现了氯霉素的分离检测。讨论了电渗流抑制剂的浓度、缓冲体系的组成和浓度等因素对检测结果的影响。线性检测范围为1~120mg/L,检出限为0.3mg/L。对市售氯霉素滴眼液和注射液中的氯霉素进行分析,加标回收率为93.8%~106.8%。     

Two-dimensional separation system of coupling capillary liquid chromatography to capillary electrophoresis for analysis of Escherichia coli metabolites

A two-dimensional (2-D) separation system of coupling chromatography to electrophoresis was developed for profiling Escherichia coli metabolites. Capillary liquid chromatography (LC) with a monolithic silica-octadecyl silica column (500 x 0.2 mm ID) was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Field-enhanced stacking was selectively employed as a concentration strategy to interface the two dimensions, which proved to be beneficial for the detection of metabolites. An artificial sample containing 118 standards, some of which lack chromophores or have weak UV absorbance, was used to optimize the 2-D separation system. Under the optimum conditions, 63 components in the artificial sample having absorbance at 254 nm could be well resolved and detected. The utility of the system was demonstrated by comprehensive analysis of E. coli metabolites. Comparing with the previous 2-D separation system we published in Anal. Chem. 2004, 76, 1419-1428, using a longer monolithic column in the first dimension improved the separation efficiency and offered the possibility of increasing the injection volume without compromising the separation efficiency. In the second dimension, field-enhanced stacking was used to improve the concentration sensitivity of the metabolites, and more metabolites in E. coli cell extract were detected and identified using the developed 2-D separation system. In addition, preliminary investigation for future CE-mass spectrometry coupling was also made in the study by using volatile buffers in the capillary LC and CE techniques.     

高效毛细管电泳-电导检测对四环素衍生物分离测定的研究

运用毛细管电泳-电导检测方法对4种四环素衍生物——土霉素(OTC)、金霉素(CTC)、强力霉素(DOC)和四环素(TC)的分离进行了研究。在3．5mmol/L三羟基氨基甲烷(Tris)-7．5mmol/L柠檬酸(Cit)pH4．0的运行缓冲液中，4种四环素衍生物在15min内获得完全分离。四环素衍生物的线性范围分别为5．0-500μg/mL OTC，3．6-420μg/mL CTC，4.5-470μg/mL DOC和2.5-400μg/mL TC。检测限(S／N＝3)分别为OTC2．0μg/mL，CTC 1．8μg／mL，DOC2．5μg/mL和TC1．0μg/mL。采用本法对实际样品强力霉素片中强力霉素和土霉素片中土霉素进行测定，回收率分别为97．2％和96.4％。     

Separation of six purine bases by capillary electrophoresis with electrochemical detection

Capillary zone electrophoresis with electrochemical detection (ED) has been employed for the separation and determination of adenine (A), guanine (G), theophylline (Thp), hypoxanthine (HX), xanthine (Xan) and uric acid (UA). Effects of several important factors such as the acidity and concentration of running buffer, separation voltage, injection time and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300 μm carbon disc electrode at a working potential of +0.95 V (versus saturated calomel electrode (SCE)). The six purine bases can be well separated within 14 min in a 40 cm length fused-silica capillary at a separation voltage of 10 kV in a 100 mmol/l borate buffer (BB, pH 10.0). The current response was linear over about three orders of magnitude with detection limits (S/N=3) ranging from 0.157×10⁻⁶ to 0.767×10⁻⁶ mol/l for all compounds. The proposed method was successfully applied to determine Thp in tea and aminophylline tablets, UA in human urine, and two purine bases in DNA.     

Signal enhancement in capillary electrophoresis by using a sleeve cell arrangement for optical detection

This study comparts signal enhancement and efficiency in CZK employing three modes of detection: the sleeve cell (a simple method for creating a region of extended path length for absorption detection), the bubble cell (extended light path capillaries), and on-column detection in 75 μm i.d. capillary. Flow profile in the sleeve cell was monitored under a microscope. An abrupt change in capillary diameter in the sleeve cell region (from 50 μm to 220 μm) did not produce extensive band broadening. The sleeve-cell detection arrangement delivered a 3.5 fold increase in corrected peak area when compared with an oil-column detection in 75 μm i.d. column.     

Detection of Escherichia coli O157:H7 DNA using two fluorescence polarization methods

Using stx 2 gene in verotoxin-producing Escherichia coli O157:H7 as a target DNA, polymerase chain reaction (PCR) amplification has been combined with fluorescence polarization (FP) by two distinct combination protocols. The first approach (PCR-probe-FP) requires that fluorescence labeled specific probes are hybridized with the asymmetric PCR product. In the second protocol (PCR-primer-FP), the fluorescence labeled primer is used in PCR amplification. In both methods, the PCR products are detected using fluorescence polarization. Hybridization (in the PCR-probe-FP method) and conversion (in the PCR-primer-FP method) of 5′-fluorescence labeled oligodeoxynucleotide to the PCR product are monitored by an increase in the anisotropy ratio. The results demonstrate the importance of asymmetric PCR (in the first method) and the selection of dye-modified primer concentration (in the second method) for designing a polarization strategy for the detection of DNA sequence. It has been found that the methods can be used for the identification of infectious agents. This system has also been applied to the determination of Escherichia coli O157:H7 strains.     

Analysis of human histone H4 by capillary electrophoresis in a pullulan-coated capillary,LC-ESI-MS and MALDI-TOF-MS

The object of the present study was the analysis of the human histone H4 (a core histone) in order to evaluate the state of its acetylation. Capillary electrophoresis (CE) using a pullulan-coated capillary provides a rapid and efficient approach to the separation of monoacetylated, diacetylated and triacetylated H4 isoforms from human cells. By using a simple running buffer of 100 mM triethanolamine-phosphate solution at pH 2.5 and exploiting the effectiveness of pullulan-based coverage in preventing adsorptive phenomena, the separation of the differently acetylated isoforms was achieved in less than 15 min with high efficiency and reproducibility. The proposed method was for the first time applied in the analysis of histone H4 fractions obtained from cell lines treated with different histone deacetylase (HDAC) inhibitors, used as potential anticancer drugs. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis demonstrated that the acetylation occurred in the histone H4 tail, whereas the CE separation allowed for a fast determination of the percentages of H4 acetylated isoforms in real samples; the results were in agreement with those obtained from liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) analysis. Therefore, the proposed CE method is a useful complementary support to the hyphenated techniques for the rapid monitoring of the activity of HDAC inhibitors.     

Detection of fuel-oxidizer explosives utilizing portable capillary electrophoresis with wipe-based sampling

Portable analytical instrumentation that can provide an alarm indication for the presence of explosives and related components is critical for the identification of explosives-based hazards and threats. Many explosives incident reports involve an inorganic oxidizer-fuel mixture which can include pyrotechnics, fireworks, flash powders, black powders, black powder substitutes, and improvised or homemade explosives. A portable CE instrument with targeted analysis of common inorganic oxidizer ions, for example, chlorate, perchlorate, and nitrate, was used here as a rapid detection platform. Unlike frequently used gas-phase separation and detection instrumentation such as ion mobility spectrometry (IMS), an automated liquid extraction mechanism is required for CE separation using acetate paper sample collection wipes. Target inorganic oxidizers were inkjet-printed onto sample wipes to investigate instrument response relative to the collected analyte spatial distribution. Overall, analyte signal intensities increased with off-center sample deposition due to improved sample extraction from wipes and no change in response was observed for varied array distributions across wipes. The system demonstrated sub 200 ng detection limits for all target analytes, with further improvement when normalizing to an internal standard.     

双通道-双工作电极毛细管电泳电化学法快速检测大黄酸

报道了一种测定大黄酸的快速毛细管电泳电化学方法。采用简易制作的一种双通道-双工作电极电化学系统,可以实现电导法和安培法同时检测;优化选择了缓冲介质、工作电极、检测电位、毛细管长度和内径以及分离电压等实验参数,并对提高分析速度进行了初步研究和探讨。结果表明:大黄酸在100s内可以得到较好的分离测定,电导法和安培法的线性范围分别为6.83×10-4～1.07×10-5mol/L和3.41×10-4～2.67×10-6mol/L,最小检出浓度分别为5.28×10-6mol/L和3.16×10-7mol/L。采用设计的双通道 双工作电极检测装置,可以充分发挥电导法和安培法的优越性,对样品峰及样品的纯度进行确证;另外通过采用较短的毛细管与适当提高分离电压,可以提高分析速度。该法已用于中药大黄中大黄酸的测定。     

未衍生化卡托普利的毛细管电泳法测定

采用毛细管电泳高频电导法测定了未衍生化的卡托普利。考察了分离检测条件的影响。实验选择10．00mmoL／L Tris-5．0mmol／LH3BO3-20．0％CH3OH为电泳介质,在优化条件下,卡托普利的线性范围为1．00μg/mL-200μg／mL,检出限为0．3μg／mL。成功地检测了血清和尿液中的卡托普利。     

毛细管电泳电化学检测法测定烟草中的多元酚

采用毛细管电泳电化学检测法同时测定了烟草中的多元酚,即芦丁、绿原酸,槲皮素和咖啡酸。考察了工作电极的氧化电位、运行缓冲溶液浓度和pH值,分离电压和进样时间对分离和检测的影响。在优化条件下,以300μm直径的碳圆盘电极为工作电极,检测电位为+0.9 V(vs.SCE),在50 mmol/L硼酸盐(pH 8.4)的运行缓冲液中,被测物浓度与峰电流在三个数量级范围内呈良好线性,检出限为2×10-7或5×10-7g/mL。方法有着良好的重现性,被测组分的迁移时间和峰高的相对标准偏差(RSDs)小于4%(n=7)。单次测定可在16 min内完成,已用于实际样品多元酚的测定,样品处理简单,无须预富集。