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1.
Gene analysis of multiple oral bacteria by the polymerase chain reaction coupled with capillary polymer electrophoresis 下载免费PDF全文
Chenchen Liu Yoshinori Yamaguchi Shinichi Sekine Yi Ni Zhenqing Li Xifang Zhu Xiaoming Dou 《Journal of separation science》2016,39(5):986-992
Capillary polymer electrophoresis is identified as a promising technology for the analysis of DNA from bacteria, virus and cell samples. In this paper, we propose an innovative capillary polymer electrophoresis protocol for the quantification of polymerase chain reaction products. The internal standard method was modified and applied to capillary polymer electrophoresis. The precision of our modified internal standard protocol was evaluated by measuring the relative standard deviation of intermediate capillary polymer electrophoresis experiments. Results showed that the relative standard deviation was reduced from 12.4–15.1 to 0.6–2.3%. Linear regression tests were also implemented to validate our protocol. The modified internal standard method showed good linearity and robust properties. Finally, the ease of our method was illustrated by analyzing a real clinical oral sample using a one‐run capillary polymer electrophoresis experiment. 相似文献
2.
Electrophoretic conditions to separate sodium dodecyl sulfate (SDS)-protein complexes according to their relative molecular mass by capillary electrophoresis (CE) using linear polyacrylamide as a sieving matrix were examined. Five purified proteins with relative molecular masses between 14 400 and 66 200 Da were separated on a coated fused-silica capillary with an internal diameter of 100 microm and an effective length of 24 cm (total length, 32.5 cm). Benzoic acid was added to the solution of purified proteins as internal standard; beta-mercaptoethanol was also added as reducing agent. The running buffer composition was 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.035 M aspartic acid, 0.1% m/v SDS, 4% m/v acrylamide, the resulting pH being 8.0. The applied voltage was 7 kV (reversed voltage polarity) in order to avoid high current intensities. Under optimized conditions, the five proteins were separated in less than 15 min, with a % relative standard deviation (RSD) between 0.2 and 0.4 for migration times in the same day. Good efficiency (values between 150 000 and 40 000 N/m) and resolution (values between 2 and 2.8) were obtained. The inverse of relative migration times was found to correlate with the logarithm of their relative molecular mass. Finally, cider proteins were analyzed and their relative molecular masses were determined. These results were compared with those obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 相似文献
3.
James P. Landers Robert P. Oda Benjamin Madden Tamara P. Sismelich Thomas C. Spelsberg 《Journal of separation science》1992,15(8):517-525
Run-to-run sample separation reproducibility has been compared on two commercial high performance capillary electrophoresis units which differ in the mode by which the capillary temperature is thermostatted. Three standard analytes, differing dramatically in molecular character and size, were used for the analysis: benzoic acid, a 14 amino acid peptide from human chorionic gonadotropin, and ribonuclease A represent, respectively, small stable organic molecules, small peptides with little or no secondary structure, and proteins with secondary structure. These standards were evaluated with regard to reproducibility of migration time, peak area, and peak height. The analyses, performed in buffers of optimum pH for the separations, demonstrated that the liquid and forced air convection thermostatted systems both performed extremely well. The reproducibility, as judged by the percent coefficient of variance (% CV) of replicate analyses, was generally found to be less than 1 % (migration time); the reproducibility decreased in the order migration time > peak height > peak area. Whereas the absolute % CV values for MTrel (migration relative to a standard) observed with the liquid thermostatted system were 2- to 4-fold lower than those observed with the forced air convection thermostatted system, there was little statistically significant difference between the two. As expected, the data indicated a reduction in reproducibility as the complexity of the analyte increased, perhaps as the result of an increased potential for wall interactions. Comparing separations in which low (≈?1 watt/meter [W/m] of capillary) and high (>5 W/m) Joule heat was generated by altering the sodium chloride content of the buffer revealed few statistically significant differences in the reproducibility obtained from the two systems. With these particular standard analytes and their respective buffer systems, there appears to be little difference between forced air convection and liquid thermostatting of the capillary. 相似文献
4.
Shi X Bányai I Lesniak WG Islam MT Országh I Balogh P Baker JR Balogh LP 《Electrophoresis》2005,26(15):2949-2959
Generation 2 to generation 5 poly(amidoamine) (PAMAM) dendrimers having different terminal functionalities were analyzed by capillary electrophoresis (CE). Polyacrylamide gel electrophoresis was also used to assess the composition of the individual generations for comparison with the CE results. Separation of PAMAMs can be accomplished by either using uncoated silica or silanized silica capillaries, although reproducibility is poor using the uncoated silica capillary. To improve run-to-run reproducibility, silanized capillary was used and various internal standards were also tested. Relative and normalized migration times of primary amine terminated PAMAM dendrimers were then determined using 2,3-diaminopyridine (2,3-DAP) as an internal standard. Using silanized capillaries and internal standards, the relative and normalized migration times are fully reproducible and comparable between runs. Apparent dimensionless electrophoretic mobilities were determined and the results were compared to theoretical calculations. It is concluded that for PAMAMs a complex separation mechanism has to be considered in CE, where the movement of the ions is due to the electric field, but the separation is rather the consequence of the adsorption/desorption equilibria on the capillary wall ("electrokinetic capillary chromatography"). The described method may be used for quality control and may serve as an effective technique to analyze polycationic PAMAM dendrimers and their derivatives with different surface modifications. 相似文献
5.
A set of 25 monoprotic bases is proposed as internal standards for pK(a) determination by capillary electrophoresis. The pK(a) of the bases is determined and compared with available literature data. The capillary electrophoresis internal standard method offers numerous advantages over other typical methods for pK(a) determination, especially of analysis time and buffer preparation. However, it requires disposing of appropriate standards with reference pK(a) value. The set of bases established in this work together with the set of acids previously established provide a reference set of compounds with well-determined acidity constants that facilitate the process of selecting appropriate internal standards for fast pK(a) determination by capillary electrophoresis in high throughput screening of pharmaceutical drugs. In addition, the performance of the method when acidic internal standards are used for the determination of acidity constants of basic internal standards has also been tested. Although higher errors may be expected in this case, good agreement is observed between determined and literature values. These results indicate that in most cases structural similarity between the analyte and the internal standard might not be an essential requirement in the internal standard method. 相似文献
6.
几种标准蛋白质的毛细管区带电泳的分离及其迁移行为 总被引:1,自引:0,他引:1
本文采用毛细管区带电泳分离了几种标准蛋白,研究了不同的清洗方式,不同PH的缓冲溶液等对迁移行为及分离的影响,并同时考察了迁移时间和相对迁移时间与操作电压的关系。 相似文献
7.
Migration-time reproducibility is essential in the use of capillary electrophoresis to identify components in mixtures. Two methods based on the migration time of either one or two reference markers are proposed for improving migration time reproducibility. These methods were evaluated to determine the migration time reproducibility for phenylthiohydantoin-amino acids, fluorescein thiohydantoin-amino acids, and tetramethylrhodamine labeled oligosaccharides. In the best case, the relative standard deviation of the migration time was reduced from >3% without correction to <0.04% with the two-marker correction. 相似文献
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A fast, simple and cost-effective one-pot labeling strategy coupled with capillary zone electrophoresis was developed for the complete separation of amino acid mixture. The strategy includes two steps of reactions: Cyanuric chloride was made to react first with 7-amino-1,3-naphthalenedisulfonic acid monopotassium salt at 0 °C for 10 min, and then with amino acids at 55 °C for 6 min. The resulted products, after diluted with water, were injected into capillary zone electrophoresis system for separation. Using a running buffer of 20 mM sodium tetraborate decahydrate at pH 10.1, nineteen amino acids were efficiently separated in 25 min, with relative standard deviation of 0.36–1.6% and 0.96–2.1% (within and between days, respectively) for migration time and 0.030–1.6% and 0.22–2.4% (within and between days, respectively) for peak area. The proposed method has been successfully applied to the determination of free amino acids in biofluids, including human serum, urine, and saliva. The linearity of quantification was over two orders of magnitude for most amino acids, with a correlation coefficient larger than 0.999. The average recovery, determined by spiking a known amount of amino acid standards into real samples, was in a range from 91.6% to 105.9%. This method can be a noninvasive means since it could directly assay the urine and saliva samples. 相似文献
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Valdemar I. Esteves Susana S.F. Lima Diana L.D. Lima Armando C. Duarte 《Analytica chimica acta》2004,513(1):163-167
The simultaneous separation and determination of organic acids in several samples of white and red Port wines was performed by capillary zone electrophoresis using indirect UV detection with 2,6-pyridinedicarboxylic acid as a background electrolyte buffer. Operational parameters like migration time, temperature, voltage and capillary length were optimized. Sixteen samples of red wine and four samples of white wine were used to analyze for tartaric, malic, lactic, succinic and acetic acids using glyoxylic acid as the internal standard. The method is rapid, sensitive and quantitative, and time-consuming sample preparation, such as solid-phase extraction or liquid-liquid extraction procedure, is not required. 相似文献
13.
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. 相似文献
14.
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. 相似文献
15.
Double‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis 下载免费PDF全文
Yi‐Wei Zhang Ming‐Zhe Zhao Jing‐Xin Liu Ying‐Lin Zhou Xin‐Xiang Zhang 《Journal of separation science》2015,38(3):475-482
Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high‐sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)‐coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high‐hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N‐glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results. 相似文献
16.
Melissa N. Dunkle 《Microchemical Journal》2006,82(1):100-107
A fluorescein-labeled estradiol derivative was assessed for use in affinity capillary electrophoresis (ACE) in a competitive immunoassay format, in which the fluorescently labeled estradiol competed with unlabeled estradiol for a mouse anti-estradiol antibody. The preparation of the labeled estradiol produced a mixture of fluorescein-containing compounds that led to multiple peaks in the electropherogram and to which the antibody responded differently. Two of the components of the mixture, towards which the mouse antibody showed most affinity, were isolated using fraction collection via capillary electrophoresis (CE). The two fractions of the labeled estradiol products isolated by CE were characterized using mass spectrometric methods. The two active fluorescein-conjugated products differed in the carboxylate on the fluorescein moiety, one having a methyl group instead of the acidic hydrogen for the other. The estradiol antibody showed a stronger binding for the conjugate containing the methyl group, as determined from the estimated binding constants using Scatchard analysis. The isolated fractions of labeled estradiol were shown to be applicable to the ACE immunoassay method. 相似文献
17.
A simple fluorescence detector for capillary electrophoresis (CE) using a blue light-emitting-diode (LED) as excitation source is constructed and evaluated. An optical fiber was used to collect the fluorescence, and a flat end of the fiber was modified to spherical end, resulting in 50% increase of efficiency over the flat end. A simple device for optical alignment of the fibers and capillary column was designed. The concentration and mass detection limits for fluorescein were 1.8×10−7 mol l−1 and 4.3 femol, respectively. 相似文献
18.
This study involves the use of polyamines as potential resolving agents for the capillary electrophoresis (CE) of glycosaminoglycans (GAGs), specifically heparin, dermatan sulfate, chondroitin sulfate, over-sulfated chondroitin sulfate (OSCS), and hyaluronan. All of the compounds can be separated from each other with the exception of chondroitin sulfate and hyaluronan. Using optimization software, the final run conditions are found to be 200 mM ethylenediamine and 45.5 mM phosphate as the electrolyte with −14 V applied across a 50 μm ID × 24.5 cm fused silica capillary at 15 °C. The ion migration order, with OSCS as the last instead of the first peak, is in contrast to previous reports using either a high molarity TRIS or lithium phosphate run buffer with narrower bore capillaries. Total analysis time is 12. 5 min and the relative standard deviation of the heparin migration time is about 2.5% (n = 5). The interaction mechanism between selected polyamines and heparin is explored using conductivity measurements in addition to CE experiments to show that an ion-pairing mechanism is likely. 相似文献
19.
This work described a sensitive method for determination of metoprolol in rabbit plasma.The method involved purification by ultrafiltration,derivatization with fluorescein isothiocyanate,determination by capillary electrophoresis(CE) coupled with laser-induced fluorescence(LIF) detector.Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol in experimental condition.The assay had a wide range(2.0-500 ng/mL) of linearity and a detection limit of 0.8 ng/mL.The intra- and inter-day precisions were satisfactory with relative standard deviation(RSD) less than 10.0%and accuracy within 10.0%.This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood. 相似文献