Use of dynamically coated capillaries with added cyclodextrins for the analysis of opium using capillary electrophoresis

A rapid, precise, accurate, and robust method using capillary electrophoresis (CE) with dynamically coated capillaries for the analysis of the major opium alkaloids in opium is presented. Dynamic coating of the capillary surface is accomplished using a commercially available reagent kit (polycation coating followed by polyanion coating). The addition of dual cyclodextrins (hydroxypropyl-beta-cyclodextrin and dimethyl-beta-cyclodextrin) to the run buffer imparts excellent selectivity for the opium alkaloids. For the determination of morphine, papaverine, codeine, noscapine and thebaine in opium gum and opium latex samples (using tetracaine as an internal standard) good agreement with values obtained by gradient high-performance liquid chromatography is obtained. Compared to the latter technique, CE affords better resolution with significantly faster analysis time (12 min versus 29 min). Dynamically coated capillaries, which give rise to a relatively high and robust electroosmotic flow (EOF) at the background electrolyte pH of 2.5, allow for rapid analysis and excellent migration time and peak area precision (RSDs < or = 0.12% and < or = 1.2%, respectively). Reproducible separations (relative migration times) for over 500 samples have been obtained on a single capillary. The nature of the injection solvent, the injection time and the contents of the waste vials have a profound effect on the pressure injection precision of the relatively hydrophobic solutes. The CE conditions reported in this study are also applicable to the analysis of lysergic acid diethylamide (LSD) exhibits.     

Separation of plant hormones from biofertilizer by capillary electrophoresis using a capillary coated dynamically with polycationic polymers.

A new, simple and rapid capillary electrophoresis (CE) method, using hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier, was developed for the identification and quantitative determination of four plant hormones, including gibberellin A3 (GA3), indole-3-acetic acid (IAA), alpha-naphthaleneacetic acid (NAA) and 4-chlorophenoxyacetic acid (4-CA). The optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.005% (w/v) of HDB. The applied voltage was -25 kV and the capillary temperature was kept constant at 25 degrees C. Salicylic acid was used as internal standard for quantification. The calibration dependencies exhibited good linearity within the ratios of the concentrations of standard samples and internal standard and the ratios of the peak areas of samples and internal standard. The correlation coefficients were from 0.9952 to 0.9997. The relative standard deviations of migration times and peak areas were < 1.93 and 6.84%, respectively. The effects of buffer pH, the concentration of HDB and the voltage on the resolution were studied systematically. By this method, the contents of plant hormone in biofertilizer were successfully determined within 7 min, with satisfactory repeatability and recovery.     

Graphene oxide coated capillary for chiral separation by CE

This article describes a new application of graphene oxide (GO) in CE based on the coating of fused silica capillary for chiral separation. The coated capillary was characterized by SEM, energy dispersive X‐ray spectroscopy, and Raman spectra. The results indicated that the capillary was coated with GO. Chiral separations were carried out in the GO‐coated capillary for the ephedrine–pseudoephedrine (E‐PE) isomers and β‐methylphenethylamine (β‐Me‐PEA) isomers at the optimal buffer conditions without any chiral selector by CE. The precision and reproducibility of GO‐coated capillary were investigated, and the RSDs of migration time (n = 6) for the E‐PE isomers were 1.35–1.41%, and 0.97–3.50% for β‐Me‐PEA isomers, respectively. The LOD for E‐PE isomers and β‐Me‐PEA isomers was 3 μM and 18 μM, respectively.     

Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary

A capillary zone electrophoresis (CZE) method was developed for the rapid analysis of charge heterogeneity of immunoglobulin G (IgG) monoclonal antibodies (mAbs). The separation was carried out in a short, dynamically coated fused-silica capillary. A number of separation parameters were investigated and optimized, including pH, concentration of the separation buffer (ε-amino caproic acid), concentration of the triethylenetetramine (TETA) dynamic coating, the capillary internal diameter and the field strength used for the separation. The effects of between-run flushing of the capillary and the data acquisition rate were also evaluated. Under the optimized conditions, a fast (<5 min), selective and reproducible separation of mAb charge variants was achieved under a very high electric field strength (1000 V/cm). This method also requires only a short conditioning of the capillary, with between-run conditioning completed within 2 min. The method was evaluated for specificity, sensitivity, linearity, accuracy and precision. The same separation conditions were applied to the rapid separation (2-5 min) of charge variants of multiple monoclonal antibodies with pI in the range of 7.0-9.5. Compared with other existing methods for charge variants analysis, this method has several advantages including a short run time, rapid capillary conditioning and simple sample preparation.     

CE-LIF analysis of mitochondria using uncoated and dynamically coated capillaries

This report is the first demonstration of the use of uncoated and dynamically coated capillaries for the separation of individual mitochondria via CE. Currently, the analysis of individual mitochondria relies upon fused-silica capillaries coated with a hydrophilic polymer (e.g. poly(acryloylaminopropanol)), which is used to minimize adsorption to the capillary surface. Both uncoated fused-silica capillaries and 0.2% w/w poly(vinyl alcohol) dynamic coating solutions are used to successfully analyze isolated individual mitochondrial particles using CE-LIF. While it was possible to separate mouse liver mitochondria on an uncoated capillary, rat liver mitochondria proved to have strong adsorption characteristics that only allowed them to be adequately separated with a PVA dynamic coating or a poly(acryloylaminopropanol) (AAP) capillary. The possible causes for this adsorption are analyzed and discussed. This study shows that uncoated and dynamically coated capillaries can be used in place of AAP-coated capillaries to analyze mitochondria and suggests the use of these capillaries for the analysis of other organelles, offering a greatly simplified method for the analysis of individual organelles.     

羧甲基壳聚糖修饰的开管毛细管电色谱柱的制备与表征

以γ-(2,3-环氧丙氧基)丙基三甲氧基硅烷为偶联剂,将羧甲基壳聚糖键合至毛细管内壁上。对毛细管预处理、硅烷化以及羧甲基壳聚糖键合反应参数进行了优化,考察了开管柱内表面结构形貌、电渗流、重现性和电色谱分离性能。扫描电镜图显示羧甲基壳聚糖均匀地键合在毛细管内表面。调节流动相的pH值可产生正向和反向电渗流,电渗流的相对标准偏差(RSD): 日内小于0.8%(n=6)、日间小于3.5%(n=3)、同一批次间小于4.3%(n=3)、不同批次柱间小于6.1%(n=3),重现性较好。4种核苷酸(腺嘌呤核苷酸、鸟嘌呤核苷酸、胞嘧啶核苷酸、尿嘧啶核苷酸)在修饰后的毛细管电色谱柱上得到了较好的分离,柱效达到36000～182000 塔板/m。结果表明,所建立的开管柱制备方法简单有效、稳定性好。     

Separation of isomers of acyl glucuronides using micellar electrokinetic capillary chromatography in dynamically coated capillaries

The acyl glucuronide metabolites of endogenous as well as of xenobiotic compounds may undergo isomerization in vitro as well as in the human body. The parent acyl glucuronide and the isomerization products may react with endogenous protein to form products which in worst cases may act as antigens and thus create an allergic response. In the present paper new methods based on micellar electrokinetic or microemulsion electrokinetic chromatography for the separation of all isomerization products as well as the hydrolysis product of acyl glucuronides are described. In order to perform the separation at lower pH values in a reasonable time dynamically coated capillaries were used. This enables the electroosmotic flow to be high and constant even at low pH. The methods were developed using S-naproxen-beta-1-O-acyl glucuronide as the model substance. The assignment of the single peaks in the electropherogram was performed tentatively based on the sequential appearance of the isomerization products with time.     

Design and evaluation of capillary electrophoresis in dynamically coated capillaries coupled with chemiluminescence detection

A dynamic coating capillary electrophoresis coupled with a simplified on-line chemiluminescence detection system was designed and evaluated. In the proposed system, poly-vinylpyrrolidone was used as dynamic coating substance in the separation buffer to reduce the unwanted protein non-specific adsorption, which was first applied in capillary electrophoresis coupling with on-line chemiluminescence detection. In order to avoid complex processing, an ordinary plastic cuvette was modified as a three-way joint. The chemiluminescence reaction conditions and capillary electrophoresis separation conditions were investigated in detail. The results showed that the coated capillary can be injected protein samples at least 30 times continuously with good repeatability. Under optimal conditions, the chemiluminescence relative intensity was linear with the concentration of hemoglobin in the range of 4-1850 μg mL(-1) and the detection limit was 2.0 μg mL(-1) (S/N=3). The relative standard deviation of migration times and peak heights for 40 μg mL(-1) hemoglobin were 2.5% and 4.1% (n=11) respectively. Interference of matrix effects was overcome by the calibration according to standard addition methods. Afterwards, the method was validated successfully and was applied to detect the concentration of hemoglobin in the serum of haemolytic patients.     

Preparation and evaluation of stable coating for capillary electrophoresis using coupled chitosan as coated modifier

A coated capillary modified with a coupled chitosan (COCH) was developed by using a simple and fast (60 min) process that could be easily automated in capillary electrophoresis instrument. The COCH coating was achieved by first attaching chitosan to the capillary inner wall, and then coupling with glutaraldehyde, and rinsing chitosan again to react with glutaraldehyde. The COCH coating was stable and showed amphoteric character over the pH range of 1.8-12.0. When the pH value was lower than 4.5, the capillary surface possessed positive charges, which caused a reversal in the direction of the electroosmotic flow (EOF). The normal EOF direction could be obtained when the pH value was higher than 4.5. The COCH coating showed strong stability against 0.1 mol/L HCl, 0.1 mol/L NaOH and other solvents compared with conventional chitosan coating. The relative standard deviation of the run-to-run, day-to-day and capillary-to-capillary coating was all below 2% for the determination of EOF. The COCH-modified capillary was applied to acidic and basic proteins analyses and high efficiency could be attained. The comparison between unmodified capillary, chitosan-modified and COCH-modified capillary for the separation of real sample, extract from Elaphglossum yoshinagae with water, was also studied. Better results could be obtained on COCH-modified capillary than the other two capillaries.     

Open-tubular capillary electrochromatography using a capillary coated with octadecylamine-capped gold nanoparticles

Octadecylamine-capped gold nanoparticles (ODA-Au-NPs) were prepared and characterized by using UV-Vis adsorption spectrum, transmission electron chromatography (TEM), SEM, and FT-IR. A simple but robust hydrophobic coating was easily developed by flushing a capillary with a solution of ODA-Au-NPs, because the positive charges were carried by the nanoparticles which strongly adsorb to the negatively charged inner surface of a fused-silica capillary via electrostatic and hydrophobic interactions. The chromatographic characteristics of the coated capillary was investigated by varying the experimental parameters such as buffer pH, buffer concentration, and percentage of organic modifier in the mobile phase. The results show that (i) resolution between thiourea and naphthalene is almost the same when comparing the electrochromatograms obtained using pH 7 buffer as mobile phase after and before the capillary column was operated using pH 11 and 3 mobile phase; (ii) no significant changes in retention time and deterioration in peak efficiency were found after 60 runs of test aromatic mixtures; and (iii) column efficiency up to 189 000 theoretical plates/meter for testosterone was obtained. All of the results indicated that the coating could act as a stable stationary phase for open tubular CEC as well as for bioanalysis.     

Fast chiral separation by ligand-exchange HPLC using a dynamically coated monolithic column

The preparation and application of dynamically coated ligand-exchange chromatography phases for enantioseparation is described. The phases were prepared by pumping a solution of N-decyl-L-4-hydroxyproline, N-hexadecyl-L-4-hydroxyproline, or N-2-hydroxydodecyl-L-4-hydroxyproline through a commercially available monolithic RP-18 column. These coatings are stable against desorption for months at ambient temperature when aqueous mobile phases are used. The columns were applied to the chiral separation of amino acids, glycyl dipeptides and diastereomeric dipeptides, and tripeptides. The chiral selector can be removed or changed easily by washing the column with ACN or methanol. Ultrafast separations in the range of seconds were achieved using high flow rates.     

Improvement of reproducibility of trace analysis by spark-source mass-spectrometry

Summary Mechanisms responsible for poor reproducibility of analysis by spark-source mass-spectroscopy are discussed. A new scheme for the output cascade of a radiofrequency generator has been developed. It allows for stabilizing both the charge and impurity mass-spectrum composition at changing the width of the interelectrode gap over a wide range. The causes of transient irreproducibility in mass-spectrometric analysis have been studied and traced to temporal variation in the discharge-circuit parameters. Methods of eliminating this irreproducibility are proposed.

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Verbesserung der Reproduzierbarkeit der funkenmassenspektrometrischen Spurenanalyse

Zusammenfassung Die Ursachen der schlechten Reproduzierbarkeit der mit Hilfe der Fun-kenmassenspektrometrie durchgeführten Spurenanalyse wurden erörtert. Ein neues Schema der Ausgangsstufe des Hochfrequenzgenerators wurde ausgearbeitet. Dies ermöglicht die Konstanthaltung sowohl der Ladung wie der Zusammensetzung des Massenspektrums auch bei weitgehender Veränderung des Elektroden-Zwischenraumes. Die Ursachen der vorübergehenden Nicht-Reproduzierbarkeit massenspektrometrischer Analysen wurden untersucht. Diese wird von der zeitlichen änderung der Ladungsparameter verursacht. Vorschläge für die Behebung dieser Nicht-Reproduzierbarkeit wurden gemacht.

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Presented at the 8th International Microchemical Symposium, Graz, August 25–30, 1980.     

Improvement of reproducibility in capillary zone electrophoresis by sequential application of high voltage and pressure

In capillary zone electrophoresis (CZE) problems arise concerning the reproducibility of the measurements. This is due to variations in electroosmotic flow and analyte velocities. The discontinuous use of high voltage for the separation of analytes and the pressure for the transport to the detector offers the possibility to suppress the main causes for these phenomena. The procedure is demonstrated for the separation of 4 phenol derivatives. An increase in reproducibility is found for standard solutions from about 3% to 1%.     

迁移时间归一化法改善毛细管电泳表征中药大黄的重现性

探讨了迁移时间归一化法改善中药毛细管电泳分析迁移时间重现性的原理,并将其应用于实际样品的分析。迁移时间归一化法认为,在相同的操作电压、缓冲液组成和温度条件下,多次电泳实验中迁移时间产生差别的主要原因是多次电泳实验中电渗流产生了差异。迁移时间归一化法就是通过选择电泳谱图中的一个或两个峰作为标记峰,将各次电泳实验的迁移时间都归一到第一次电泳实验中的迁移时间。比较多次电泳实验中迁移时间(t)的相对标准偏差(RSD)、经单峰归一化处理的迁移时间(t′)的RSD、经双峰归一化处理的迁移时间(t″)的RSD、迁移时间比(t/tistd,istd代表所选择的标记物)的RSD,发现RSD(t″)相似文献   

CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection

The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.     

Metal adsorption of carboxymethyl cellulose/carboxymethyl chitosan blend hydrogels prepared by Gamma irradiation

Blend hydrogels based on the carboxymethyl cellulose (CMC) and carboxymethyl chitosan (CMCts) were prepared by γ-irradiation of a high concentrated CMC/CMCts aqueous solution. Properties of the hydrogels, such as gel fraction, swelling ratio, gel strength, and metal adsorption for Pb and Au were investigated. The gel fraction increased with increasing dose, while the swelling ratio decreased with increasing it. The obtained blend hydrogels had high adsorption performance which was controlled by adjusting the composition of CMC/CMCts.     

Open-tubular gas chromatography using capillary coated with octadecylamine-capped gold nanoparticles

The octadecylamine-capped gold nanoparticles (ODA-Au-NPs) were prepared and directly used to coat the capillary wall. The hydrophobic coating acted as the stationary phase for open-tubular gas chromatography (OTGC). The ODA-Au-NPs can be adsorbed tightly onto the inner surface of fused silica capillary column via electrostatic interaction and enhanced interaction of van der Waals between gold nanoparticles and the capillary wall. Thus, the modification of the inner surface of capillary column by ODA-Au-NPs can be achieved simply by flushing the capillary with a solution of ODA-Au-NPs and the resulted ODA-Au-NPs coating is very stable. No perceptible degradation in the ODA-Au-NPs-based separation was observed after ∼1900 sample runs. This type of columns also provided excellent chromatographic performances: high number of theoretical plates, outstanding run-to-run and column-to-column reproducibility, and high selectivity for a wide range of test mixtures. An efficiency of 2474 theoretical plates per meter for chlorobenzene was obtained on an ODA-Au-NPs-modified 1.6 m × 100 μm i.d. fused silica capillary column.     

Zwitterionic ion chromatography using a dynamically coated column and mobile phase recycling

An investigation into the use of zwitterionic ion chromatography for the determination of inorganic anions in water samples was carried out. When using an ODS stationary phase precoated with Zwittergent 3-14 and a pure water mobile phase, the stability of the adsorbed coating was insufficient for quantitative work. Recycling of the water mobile phase was used to stabilise the zwitterionic coating, and resulted in improved retention time precision (15.2% RSD down to 2.4% RSD for nitrate). Post-detection cation- and anion-exchange columns in acid and hydroxide form removed sample ions from the recycling mobile phase, with the desorbed Zwittergent 3-14 passing through unretained and passing back through the pump to the analytical column. A 200-ml volume of mobile phase was recycled over a 3-week period with retention times for sulphate, chloride and nitrate standards injected at the start and end of the period varying less than 2.5%. The same system was then used with a mobile phase containing 2 mM Zwittergent 3-14. This resulted in further improvements in retention time (0.2-0.5% RSD, n = 10) and peak area precision (2.6-6.0% RSD, 1 mM standards) and improved peak efficiencies (2421-4047 N). The developed method was applied to water samples, and results compared to those obtained using anion-exchange chromatography. All sample cations were exchanged to sodium using an off-line cation-exchange procedure prior to injection.     

Determination of dissociation constants of aristolochic acid I and II by capillary electrophoresis with carboxymethyl chitosan-coated capillary

Aristolochic acid-I and aristolochic acid-II have been proved to be the main bioactive and toxic component in Aristolochia plants. As a result, the determination of their dissociation constants, which are important property parameters for weak acids, is highly desired for related pharmacological and toxicological studies. In this work, the dissociation constant values of aristolochic acid-I and aristolochic acid-II were determined by capillary electrophoresis using carboxymethyl chitosan-coated capillary, based on their electrophoretic mobilities by using nonlinear regression as well as linear regression, showing that the two models give comparable results. The data were also compared with those obtained by capillary electrophoresis with polybrene-coated capillary, and no conspicuous difference was observed. The correlation coefficients were all higher than 0.998 for both linear and nonlinear regression model. The pKa values were found to be 3.3±0.1 for aristolochic acid-I and 3.2±0.1 for aristolochic acid-II.