毛细管等电聚焦和电渗泵驱动聚焦区带分离蛋白质

建立了一种利用电渗泵驱动毛细管内的聚焦区带,实现毛细管电泳等电聚焦分离蛋白质的方法。通过控制电压来调节泵的输出流量,从而调节聚焦区带的迁移速度。适用于毛细管电泳等电聚焦两步法分离蛋白质等两性物质。考察了对牛血清白蛋白和溶菌酶两种粗提蛋白质混合物的分离,迁移时间的RSD分别为1.6%和1.3%,峰面积的RSD均为1.6%,证明方法可行。     

毛细管电泳法对南方水牛奶乳清蛋白的分离和定量分析

利用毛细管区带电泳对广东省水牛乳乳清蛋白成分进行了分离和定量分析研究.采用1.2%的十四水合硼酸钠电泳缓冲液,对水牛奶乳清蛋白的四种主要组分α-乳白蛋白(α-La)、β-乳球蛋白(β-Lg)、牛血清白蛋白(BSA)、免疫球蛋白(IgG)进行了很好的分离,其迁移时间和峰面积的RSD分别小于1.5%和0.5%,加标回收率范围91%～102%.建立了基于毛细管区带电泳的分析方法,对牛乳及其乳制品中的乳清蛋白进行了快速分离和定量分析.     

毛细管电泳-电化学发光法测定石蒜鳞茎中的石蒜碱

建立了毛细管电泳-电化学发光(CE-ECL)检测石蒜碱的方法。探讨了检测电位、分离缓冲液pH和浓度、分离电压、进样电压及进样时间等因素对石蒜碱分离检测的影响。在最佳实验条件下,石蒜碱含量在0.03~2μg/mL范围内与其峰面积呈良好的线性关系,相关系数为0.9995,检出限为0.005μg/mL(S/N=3)。对0.8μg/mL石蒜碱进行7次平行实验,其迁移时间、峰高和峰面积的RSD分别为1.7%,5.2%和5.6%。方法应用于石蒜鳞茎中石蒜碱的测定,加标回收率为92.2%~98.6%。     

毛细管电泳-柱端喷壁式电化学检测法用于利尿剂氢氯噻嗪和氨苯喋啶的研究

应用毛细管电泳-电化学检测法对利尿剂氢氯噻嗪和氨苯喋啶进行了研究。考察了电化学检测和电泳分离条件对氢氯噻嗪和氨苯喋啶分离、检测的影响,结果表明在最佳分离、检测条件下,两种待测物在8 min内达到基线分离。氨苯喋啶和氢氯噻嗪的检测限分别达到0.29和0.25 mg/L。对两物质于日内和日间重复测定7次,迁移时间的日内相对标准偏差(RSD)不大于1.6％,峰电流的日内RSD不大于 3.1％; 迁移时间的日间RSD不大于1.7％,峰电流的日间RSD不大于 4.9％。 将该方法用于复方氨苯喋啶成药中氨苯喋啶和氢氯噻嗪的分离和测定,成药的检测结果与标示量比较,相对误差小于4.6％。在模拟尿样中对氢氯噻嗪和氨苯喋啶进行标准溶液添加回收实验,其回收率分别为93.5%~96.7%和96.6%~97.2%,结果令人满意。     

高效毛细管电泳间接紫外检测血浆中钾、钙、镁

用高效毛细管电泳法,在pH 5.5的缓冲介质中用酒石酸作为络合剂,使血浆中钾、钠、钙、镁4种阳离子达到很好分离,用咪唑作为背景试剂进行了紫外检测.对电泳分离及紫外检测的各分析条件(包括背景缓冲溶液的pH值,咪唑溶液的浓度及酒石酸溶液的浓度等)作了试验并予以优化,上述4种离子的相互分离在4.5 min内顺利完成.文中给出了K 、Ca 、Mg2 的线性回归方程,其相关系数在0.998 4～0.999 4之间,证明了在各离子的峰面积与其浓度之间呈线性关系,3种离子的检出限(S/N=3)依次为0.20,0.12,0.06 mg·L-1.用迁移时间检测和用峰面积检测所得的RSD值(n=6)依次小于0.76%和2.83%.由于钠离子的吸收峰与系统峰重叠,钠的测定无法进行.回收率试验的结果在95.3%至104.2%.     

红景天中红景天甙和酪醇的毛细管电泳法分析

利用毛细管电泳法分离测定两种红景天中红景天甙和酪醇的含量,所用毛细管规格为48.5cm×50μm,二极管阵列紫外检测器(DAD)检测波长221nm,最佳分离条件:电压21kV,分离温度25℃,背景电解质为含有30mmol/L十二烷基硫酸钠(SDS),2.5+97.5(V/V)乙腈的14mmol/L硼酸溶液,pH10.7。红景天甙与酪醇分别在60.0～7.5μg/mL和27.5～3.5μg/mL质量浓度范围内与电泳峰面积呈现良好线性关系,检测下限分别为3.0和1.5μg/mL。对标准品进行6次测定,迁移时间的RSD为0 25%和0 39%,峰面积的RSD为5 26%和3 52%。     

毛细管区带电泳高效分离和高灵敏检测α-乳清蛋白和β-乳球蛋白

α-乳清蛋白和β-乳球蛋白系由牛乳清提取的乳糖制品中的主要蛋白杂质,是引起食物及药物过敏的主要过敏原。因此,在食品安全及医药安全领域,对二者的分离及痕量检测是至关重要的。本研究建立了一种简单、快速、灵敏、重现性好的毛细管区带电泳(CZE)方法,使用简单的电泳体系和样品处理步骤,实现了α-乳清蛋白和β-乳球蛋白的基线分离和痕量检测。CZE条件如下:25 mmol/L磷酸盐缓冲液(pH 7.0),分析电压+30 kV,毛细管温度25℃,紫外检测波长205 nm,压力进样5 kPa"10 s。在此条件下,样品分析时间为2 min,α-乳清蛋白和β-乳球蛋白的检出限(LOD)分别为3.0和12 mg/L;迁移时间和峰面积的相对标准偏差(RSD,n=6)分别小于1%和6%,符合实际样品检测要求。本方法已成功用于实际乳糖样品的分析,在相关领域也有很好的应用前景。     

毛细管电泳法分析唐古特白刺种子中两种生物碱

利用毛细管电泳法分离测定唐古特白刺种子中的尿囊素和吲哚生物碱1-methyl-1,2,3,4-tetrahydro--βcarboline-3-carboxylic acid(MTCCA),所用毛细管规格为48.5 cm×50μm i.d.,DAD检测波长220 nm,最佳分离条件:电压19 kV,分离温度25℃,背景电解质为含有32 mmol/L SDS,体积分数10.0%乙腈的32mmol/L硼酸溶液,pH 10.0。MTCCA与尿囊素分别在350.0~11.0μg/mL和112.5~3.5μg/mL质量浓度范围内与电泳峰面积呈现良好线性关系,检出限分别为5.0μg/mL和2.5μg/mL。对标准品进行6次测定,迁移时间的RSD为1.1%和1.4%,峰面积的RSD为2.3%和0.82%。     

苯胺类化合物的胶束电动毛细管色谱优化分离

研究了用胶束电动毛细管色谱分离对-氯苯胺、对-溴苯胺、2,4-二氯苯胺、对-碘苯胺;在选定的实验条件下,各化合物迁移时间的相对偏差(RSD)＜0.5%,峰面积RSD＜3.0%,苯胺化合物的检出限为(1.2～4.2)×10-6mol/L;方法用于实验室废水分析,结果令人满意.     

毛细管电泳-电致化学发光法分离测定麻黄中的麻黄碱、伪麻黄碱与甲基麻黄碱

以PB-Eu化学修饰电极为工作电极,对乙烯基苄基三乙基氯化铵离子液体为拆分添加剂,首次采用毛细管电泳-电致化学发光法对麻黄碱、伪麻黄碱和甲基麻黄碱进行了分离和检测。考察了检测电位、分离缓冲液的种类和酸度、添加剂用量等条件对电泳分离效果及检测灵敏度的影响。在优化条件下,3种混合药物可在8 min内达到基线分离,甲基麻黄碱、麻黄碱和伪麻黄碱的质量浓度分别在0.025～10、0.025～25、0.05～10 mg/L范围内与其峰面积呈良好但斜率略不同的两区段型线性关系,总的线性响应范围可达3个数量级。以质量浓度均为1.00 mg/L的3种混合药物合成样品进行6次平行测试,其峰面积和迁移时间的RSD分别小于4.5%和0.95%。该方法成功用于商品麻黄碱类药物制剂及中药麻黄原药中3种生物碱含量的测定,加标回收率为101%～111%。     

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.     

In-capillary non-covalent labeling and determination of tomato systemin with quantum dots in capillary electrophoresis with laser-induced fluorescence detection

Quantum dots (QDs), with their superior size-dependent fluorescence properties, have been employed as non-covalent fluorescent labels for the determination of tomato systemin (TomSys) by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The optimum conditions of in-capillary labeling and CE separation were investigated in detail, and complete separation of QDs-labeled TomSys from free QDs labels was achieved. Satisfactory results were obtained in terms of linearity (R(2)=0.998), sensitivity (limit of detection, 66 fmol) and repeatability (run-to-run RSDs of migration time and peak area, 0.9 and 4.6%, respectively; day-to-day RSDs of migration time and peak area, 3.1 and 11.9%, respectively). The established CE-LIF method was later applied in the detection of TomSys spiked in the sample of tomato leaves, which showed the applicability of the proposed method in the analysis of the target plant peptide hormone in the complex matrix.     

In-capillary solid-phase extraction-capillary electrophoresis for the determination of chlorophenols in water

A novel CE method combined with SPE in a single capillary was developed for analysis of chlorophenols in water. A frit of 0.5 mm was first made by a sol-gel method, followed by packing a SPE sorbent in the inlet end of the capillary. Two phenol derivatives, 2,4-dichlorophenol and 2,4,5-trichlorophenol, were used as the model compounds. By loading sample solutions into the capillary, the two chlorophenols were extracted into the sorbent. They were desorbed by injecting only about 4 nL of methanol. Finally, the analytes were separated by conventional CE. The technique provided a concentration enhancement factor of over 4000-fold for both chlorophenols. The detection limits (S/N = 3) of 2,4-dichlorophenol and 2,4,5-trichlorophenol were determined to be 0.1 ng/mL and 0.07 ng/mL, respectively. For replicate analyses of 5 ng/mL of 2,4-dichlorophenol, within-day and between-day RSDs of migration time, peak height and peak area were in the range of 1.8-2.0%, 4.0-4.4% and 4.1-4.6%, respectively. The method shows wide linear range, acceptable reproducibility and excellent sensitivity, and it was applied to the analyses of spiked river water samples. The capillary packed with the SPE sorbents can be used for more than 400 runs without performance deterioration.     

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.     

Fast determination of paracetamol and its hydrolytic degradation product p-aminophenol by capillary and microchip electrophoresis with contactless conductivity detection

Paracetamol (PAC) is one of the most extensively used analgesics and antipyretic drugs to treat mild and moderate pain. P-aminophenol (PAP), the main hydrolytic degradation product of PAC, can be found in environmental water. Recently, CE has been developed for the detection of a wide variety of chemical substances. The purpose of this study is to develop a simple and fast method for the detection and separation of PAC and its main hydrolysis product PAP using CE and microchip electrophoresis with capacitively coupled contactless conductivity detection. The determination of these compounds using microchip electrophoresis with capacitively coupled contactless conductivity detection is being reported for the first time. The separation was run for all analytes using a BGE (20 mM β-alanine, pH 11) containing 14% (v/v) methanol. The RSDs obtained for migration time were less than 4%, and RSDs obtained for peak area were less than 7%. The detection limits (S/N = 3) that were achieved ranged from 0.3 to 0.6 mg/L without sample preconcentration. The presented method showed rapid analysis time (less than 1 min), high efficiency and precision, low cost, and a significant decrease in the consumption of reagents. The microchip system has proved to be an excellent analytical technique for fast and reliable environmental applications.     

Simultaneous determination of low-molecular-weight organic acids and chlorinated acid herbicides in environmental water by a portable CE system with contactless conductivity detection

This report describes a method to simultaneously determine 11 low-molecular-weight (LMW) organic acids and 16 chlorinated acid herbicides within a single run by a portable CE system with contactless conductivity detection (CCD) in a poly(vinyl alcohol) (PVA)-coated capillary. Under the optimized condition, the LODs of CE-CCD ranged from 0.056 to 0.270 ppm, which were better than for indirect UV (IUV) detection of the 11 LMW organic acids or UV detection of the 16 chlorinated acid herbicides. Combined with an on-line field-amplified sample stacking (FASS) procedure, sensitivity enhancement of 632- to 1078-fold was achieved, with satisfactory reproducibility (RSDs of migration times less than 2.2%, and RSDs of peak areas less than 5.1%). The FASS-CE-CCD method was successfully applied to determine the two groups of acidic pollutants in two kinds of environmental water samples. The portable CE-CCD system shows advantages such as simplicity, cost effectiveness, and miniaturization. Therefore, the method presented in this report has great potential for onsite analysis of various pollutants at the trace level.     

Quick stabilization of capillary for rapid determination of potassium ions in the blood of epilepsy patients by capillary electrophoresis without sample pretreatment

Mutations in the potassium channel genes may be linked to the development of epilepsy and affect the blood potassium levels. Therefore, accurate determination of potassium in the blood will be critical to diagnose the cause of epilepsy. CE is a competent technique for the fast detection of multiple ions, but complicated matrices of a blood sample may cause significant variation of migration times and the peak shape. In this work, a procedure for rapid stabilization of the capillary inner surface through preflushing of a blood sample was employed. The process takes only 40 min for a capillary and then it can be used for more than 2 weeks. No pretreatment of the blood sample or other surface modification of the capillary is needed for the analysis. The RSDs of the migration time and peak area were reduced to 1.5 and 5.1% from 12.6 and 14.5%, respectively. The proposed method has been successfully applied to the determination of the potassium contents in the blood sample of patients with epilepsy at different stages. The recoveries of potassium ions in these blood samples are in a range from 86.5 to 104.5%.     

Determination of poly-beta-hydroxybutyric acid in Bacillus thuringiensis by capillary zone electrophoresis with indirect ultraviolet absorbance detection

A new capillary electrophoresis method for determining poly-beta-hydroxybutyric acid (PHB) in Bacillus thuringiensis was established. Poly-beta-hydroxybutyric acid in samples was hydrolyzed by sulphuric acid and neutralized by Ba(OH)2. The content of produced beta-hydroxybutyrate was then determined by capillary zone electrophoresis (CZE) with indirect UV detection at 254 nm. With 5 mM p-hydroxybenzoate and 0.5 mM tetradecyltrimethylammonium bromide (TTAB) at pH 8.0 as carrier electrolyte, beta-hydroxybutyrate can be determined within 6 min. Standard regression equation was made by beta-hydroxybutyrate, and the linear range was 2-1000 microg/ml. The relative standard deviations (RSDs) for migration time and peak area are both less than 1.0%. The detection limit for beta-hydroxybutyrate was 0.2 microg/ml, which is two to three orders of magnitude lower than that of the gas chromatography (GC) method. The capillary electrophoresis method was successfully applied to determine poly-beta-hydroxybutyric acid in fermentation broth and single colony. The added standard recovery was 96%.     

Sensitive detection of tumor marker CA15-3 in human serum by capillary electrophoretic immunoassay with chemiluminescence detection

A new and sensitive non-competitive immunoassay (IA) for tumor marker carbohydrate antigen 15-3 (CA15-3) by CE coupling with ECL detection has been developed. This method is based on luminol-H(2)O(2 )reaction catalyzed by horseradish peroxidase (HRP). The optimum CE separation and CL detection conditions were investigated. After the non-competitive immunoreaction, the free HRP-labeled CA15-3 antibody (Ab*) and the bound Ab*-antigen (Ab*-Ag) complex were separated in a separation capillary and then catalyzed the CL reaction of luminol and H(2)O(2 )in a reaction capillary following the separation capillary. The calibration curve based on the peak areas of Ab*-Ag complex plotted against the concentrations of CA15-3 is in the range of 0-250 U/mL with a correlation coefficient of 0.9983 and the detection limit is 0.035 U/mL (S/N = 3). The response for five consecutive injections of 125 U/mL CA15-3 resulted in RSDs of 0.83% and 3.1% for the migration time and the peak area, respectively. The method was successfully used for the quantification of CA15-3 in human sera obtained from healthy persons and from patients with breast cancer.     

Determination of vitamin C and preservatives in beverages by conventional capillary electrophoresis and microchip electrophoresis with capacitively coupled contactless conductivity detection

The separation and detection of commonly used preservatives (benzoate, sorbate) and vitamin C by both conventional CE and microchip electrophoresis with capacitively coupled contactless conductivity detection is presented. The separation was optimized by adjusting the pH-value of the buffer and the use of hydroxypropyl-beta-CD (HP-beta-CD) and CTAB as additives. For conventional CE, optimal separation conditions were achieved in a histidine/tartrate buffer at pH 6.5, containing 0.025% HP-beta-CD and 0.1 mM CTAB. LOD ranged from 0.5 to 3 mg/L (S/N = 3) and the RSDs for migration time and peak area were less than 0.1 and 2%, respectively. A considerable reduction of analysis time can be accomplished by using microchip electrophoresis without significant loss in sensitivity under optimal separation conditions. A histidine/tartrate buffer at pH 6.5, incorporating 0.06% HP-beta-CD and 0.25 mM CTAB, gave detection limits ranging between 3 and 10 mg/L and satisfactory reproducibilities of < or =0.4% for the migration time and < or =3.5% for the peak area. The methods developed are useful for the quantitative determination of food additives in real samples such as soft drinks and vitamin C tablets.