Isotachophoresis-based sample preparation of cellulases in sugarcane juice using bovine serum albumin as a model protein

The foremost requirement of quantification of cellulases expressed in genetically modified sugarcane is an efficient sample clean-up. This work investigates the feasibility of isotachophoresis for this purpose. An electrolyte system comprising a leading electrolyte of 10 mM formic acid at pH 9.0 and a terminating electrolyte of 10 mM β-alanine was devised and used to perform isotachophoresis of cellulases. The use of a simple front cutting method removed a majority of interfering species in the juice, thereby resulting in the formation of a distinct zone of desired proteins. In comparison to techniques such as ultrafiltration and liming, the analysis time and loss of desired proteins was lower when the sample was prepared by using isotachophoresis. Hence, isotachophoresis was an ideal choice for purification of the proteins in question from the remaining components in the juice.     

Determination of halofuginone in feedstuffs by the combination of capillary isotachophoresis and capillary zone electrophoresis in a column-switching system

A method has been developed for the determination of the coccidiocidic drug halofuginone in feedstuff concentrates which is based on the combination of capillary isotachophoresis and capillary zone electrophoresis in the column-switching mode. The high load capacity of the isotachophoretic step and high sensitivity of the zone electrophoretic step enabled analysis of up to 25 microliters of sample solution containing as little as 10(-8) M halofuginone with excellent reproducibility (R.S.D. about 1%). Attention was paid to the possibility of the existence of transient local isotachophoresis in the zone electrophoretic step, and experimental and theoretical methods of revealing zones migrating isotachophoretically in the background electrolyte were shown.     

Determination of histamine in biological fluids by capillary isotachophoresis and fluorescence

Analytical isotachophoresis was used for the determination of histamine in biological fluids. For biological fluids with a very low content of histamine a method was developed that combines the fluorescing condensation product of histamine and o-phthaldialdehyde with the high concentration effect of isotachophoresis. This method permits the determination of histamine in serum and other biological fluids down to less than 3 ng/ml.     

Evaluation of isotachophoresis as a method of identification and comparison with other methods by information theory

Information theory is applied to estimate the identification power of isotachophoresis. Two approaches are used to estimate the information content of qualitative isotachophoresis. In the first, an expression is derived for the maximum number of compounds that can be distinguished in an isotachopherogram under realistic conditions; if equal probabilities of the incidence of the compounds are assumed, the maximum information content is 6.5–7 bit. In the second approach, the information content obtained by a retrieval procedure from a library of data on 263 anionic species in eight leading electrolyte systems in the pH range 3–10 is discussed; the information content is 3.5–5.7 bit for the individual electrolyte systems. When correlation between the systems is taken into account, the information content is reduced, e.g., to an average of 3.2 bit per system for the four systems with lowest correlation. Based on the information content, isotachophoresis is compared with mass spectrometry, infrared spectrometry, gas chromatography and thin-layer chromatography. It is shown that the identification power of isotachophoresis is in the range of that of packed-column gas chromatography.     

电堆积与等速电泳结合的毛细管电泳进样富集方法的研究

 提出电堆积与等速电泳结合的毛细管电泳进样富集方法 ,并对电堆积和等速电泳进样富集的最佳条件进行了研究。在 15kV电压 ,电堆积进样 70s和等速电泳富集 40s条件下 ,对两种结构相近的药物普萘洛尔和美托洛尔用毛细管区带电泳法进行了分离。pH 4.0 ,30mmol/L醋酸钠 醋酸、30mmol/Lβ 丙氨酸 醋酸和 1.5 mmol/L醋酸钠 醋酸溶液分别作背景 (或前导 )、尾随和样品缓冲液。与常规电迁移进样方法比较 ,信号增强因子约为 2 5 0和16 0 ;总分析时间与常规法相近。     

Determination of the ascorbate content of photographic developer solutions using miniaturised isotachophoresis on a planar chip

The use of miniaturised isotachophoresis, performed on a planar poly(methyl methacrylate) device with integrated platinum conductivity electrodes, for the analysis of the ascorbate content of photographic developer solutions has been investigated. An electrolyte system has been developed which enabled the analysis to be made without interference from any of the other components in the developer solution, a number of which were present at significantly higher concentrations than that of the ascorbate ions. Using this system, the ascorbate content of the developer solutions could be analysed in under 6 min. The limit of detection for ascorbate using miniaturised isotachophoresis was calculated to be 0.011 mmol dm(-3).     

Pretreatment of body fluids by preparative isotachophoresis prior to chromatographic analysis

Summary This study focusses attention on the possibilities of preparative isotachophoresis (ITP) as a sample pretreatment technique prior to liquid chromatographic (HPLC) analysis. The increased demand for accurate and less time consuming analysis necessitates that sample pretreatment procedures, should be develop in parallel with other improvements (e. g. in detection and separation) which can be observed. The preparation isotachophoresis was performed on gel slabs and the zones of interest were subsequently cut out, desorbed and the desorbates analyzed by HPLC. In this study satisfactory recoveries of between 85–90% with a standard deviation of 1–5% were observed for blank experiments. For spiked serum and urine samples the recoveries in general decreased with decreasing spiked drug concentrations. These observations are discussed in this paper.     

Transient pseudo-isotachophoresis for sample concentration in capillary electrophoresis

We show that many water miscible organic solvents such as acetonitrile, acetone and small alcohols can function as a terminating ion in transient isotachophoresis, which leads to sample concentration on the capillary. It is suggested that this method could be termed transient "pseudo-isotachophoresis" (pseudo-ITP). Because of their low conductivity, these water miscible organic solvents provide the high field strength necessary for band sharpening similar to that provided by the terminating ion. Salts, when present in such samples act briefly as leading ions, migrating rapidly in the organic solvent until they are slowed at the interface of the separation buffer. When the organic solvents are added to the sample, both the migrations as well as the stacking of the analytes are affected by the concentration of salts (leading ions) in the sample, similar to that observed in isotachophoresis. Our results show that this type of stacking offers good reproducibility and reliability for practical analysis. In practice, pseudo-ITP stacking is much easier to perform compared to that of true ITP with several added practical advantages as discussed.     

毛细管电泳中样品在线预富集方法

本文对高效毛细管电泳中样品在线预富集方法作了评述，包括瞬间等速电泳，反流速电泳，场放大进样，场放大排除基体和固相萃取等技术，引用文献81篇。     

Capillary zone electrophoresis separations of enantiomers present in complex ionic matrices with on-line isotachophoretic sample pretreatment.

Analytical capabilities of capillary zone electrophoresis (CZE) with on-line coupled capillary isotachophoresis (ITP) sample pretreatment in the column-coupling capillary electrophoresis equipment to separate and determine enantiomers present in multicomponent ionic matrices were studied. Tryptophan was used as a model analyte in the ITP-capillary zone electrophoresis experiments performed in this context while a 90-component model mixture of UV-light absorbing organic anions and urine served as multicomponent sample matrices. Various working modes in which the on-line coupled capillary isotachophoresis-capillary zone electrophoresis combination in the column-coupling separation system can operate were employed in the anionic regime of the separation with direct injections of the samples. Advantages and limitations of these working modes in the separations of enantiomers present in model and urine matrices were assessed. Experiments with model mixtures of tryptophan enantiomers revealed that the two were resolved in the capillary zone electrophoresis stage with the aid of alpha-cyclodextrin also when their concentration ratio in the sample was 1:200 while the concentration of L(-)-tryptophan was 25 nmol/l. The limits of detection for the enantiomers were at approximately 10 nmol/l (approximately 1.5 ng/ml) concentrations for a 220 nm detection wavelength of the UV detector employed in the capillary zone electrophoresis stage and for a 30 microliters sample load. A high sample load capacity of the on-line coupled capillary isotachophoresis stage was effective in separating the samples corresponding to 3-6 microliters volumes of undiluted urine. The results from the runs with urine samples showed that only the capillary isotachophoresis-capillary zone electrophoresis combination with a post-column on-line coupled capillary isotachophoresis sample clean-up (responsible for a removal of more than 99% of the sample anionic constituents migrating in the on-line coupled capillary isotachophoresis stack and detectable in the capillary zone electrophoresis stage) provided a universal alternative for the detection and quantitation of the model analyte (L(-)-tryptophan).     

On-line preconcentration and quantitative analysis of peptide hormone of brain and intestine using on-column transient isotachophoresis coupled with capillary electrophoresis/electrospray ionization mass spectrometry

A new approach was described to achieve very sensitive analysis of peptide hormone of brain and intestine by capillary electrophoresis coupled with a transient isotachophoresis (tITP) preconcentration method. The system used was electrospray ionization mass spectrometry (ESI-MS) as detector and equipped with a sheath flow configuration. The effects of sample matrix, pH and concentration of leading electrolyte (LE), sample injection time, and ESI-MS instrumental parameters on the efficiency of the sample stacking were investigated in detail. Under the optimized conditions, lower than micromole (0.01 microM) concentrations of the peptides were easily detected. Compared to traditional hydrodynamic injection methods, about 40-230-fold increase in detection sensitivity was obtained by this technique. A distinguishing feature of the described approach is that the background electrolyte can serve as terminating electrolyte (TE), which simplifies the process of the experiments. The method was further evaluated by the analysis of low concentration active peptide mixtures spiked in hypothalamus tissue of the rat.     

Isotachophoretic analysis of pharmaceutic formulations with different matrices: Determination of nicotinic acid in capsules,tablets, dragees,ointments and tinctures

The selectivity of isotachophoresis is investigated concerning the quantitative determination of an anionic agent in a variety of pharmaceutic application forms with different matrices. As a result, no interferences caused by matrix components were observed in any case. Therefore sample pretreatment can be neglected. The simultaneous quantitation of other anionic constituents, present in the formulations, is possible. The method is evaluated by other analytical characteristics besides selectivity, like precision, accuracy and time of analysis. The contributions of aliquotation and isotachophoretic measurement to the total error were determined by analysis of variance. Systematic errors, due to adsorption of the analyte on solid matrix material were examined by standard addition. The results of the analytical characteristics indicate the excellent applicability of isotachophoresis for the routine analysis of anionic analytes in pharmaceutic preparations     

Determination of impurities and counterions of pharmaceuticals by capillary electromigration methods

The review presents a survey of recent applications of high‐performance capillary electromigration methods—capillary zone electrophoresis, nonaqueous capillary electrophoresis, capillary isotachophoresis, micellar electrokinetic chromatography, microemulsion electrokinetic chromatography and capillary electrochromatography—for the determination of impurities of pharmaceuticals, including chiral impurities, for the period 2007–2013. In addition, due to the missing evaluation of the determination of counterions of pharmaceuticals by capillary electromigration methods in the last 20 years, the publications dealing with this topic since 1995 are included in this review. General aspects of both these types of applications of capillary electromigration methods in pharmaceutical analysis are discussed, and detailed experimental conditions used for determination of various chemical impurities and counterions of many particular drugs are described.     

Comparison of different conductivity detector geometries on an isotachophoresis PMMA-microchip

Conductivity detection is one of the most often employed means of detection in isotachophoresis. In microanalytical devices, thin-film platinum electrodes can be used for conductivity detection and for other electrochemical methods of detection. The design and the performance of different electrode geometries for on-column contact conductivity detection with thin-film platinum electrodes integrated on an isotachophoresis PMMA-microchip is described. Three different electrode geometries for direct conductivity detection were used for the investigation of isotachophoretic separations. The influence of the width of the electrodes and their positioning relative to the separation channel was investigated. The performance of the different detectors is compared for the analysis of organic carboxylic acid anions.     

Determination of phytic acid and inositolphosphates in barley

Phytic acid (PA) and lower inositolphosphates (InsP(n) ) is the main storage form of phosphorus in grains or seeds. The content of PA and InsP(n) in different varieties of barley was analyzed by capillary isotachophoresis and online-coupled capillary isotachophoresis with CZE. The electrolytes (in demineralized water) for the isotachophoretic analysis consisted of 10?mM HCl, 14?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (leading) and 10?mM citric acid (terminating). The optimized electrolytes for the online coupling isotachophoresis with zone electrophoresis analysis were mixtures of 5?mM HCl, 7?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (leading), 20?mM citric acid, 10?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (background) and 10?mM citric acid (terminating). PA and all studied InsP(n) were separated within 25?min and detected by a conductivity detector. Simple sample preparation (acidic extraction), sufficient sensitivity, speed of analysis, and low running cost are important attributes of the electrophoretic methods. The method was used for the determination of PA and InsP(n) in barley varieties within an ongoing research project.     

Rapid quantification of quinine by multi‐stacking in a portable microchip electrophoresis system

A new multi‐stacking pre‐concentration procedure based on field‐enhanced sample injection (FESI), field‐amplified sample stacking, and transient isotachophoresis was developed and implemented in a compact microchip electrophoresis (MCE) with a double T‐junction glass chip, coupled with an on‐chip capacitively coupled contactless conductivity detection (C⁴D) system. A mixture of the cationic target analyte and the terminating electrolyte (TE) from the two sample reservoirs was injected under FESI conditions within the two sample‐loading channels. At the double T‐junction, the stacked analyte zones were further concentrated under field‐amplified stacking conditions and then subsequently focused by transient‐isotachophoresis and separated along the separation channels. The proposed multi‐stacking strategy was verified under a Universal Serial Bus (USB) fluorescence microscope employing Rhodamine 6G as the model analyte. This developed approach was subsequently used to monitor the target quinine present in human plasma samples. The total analysis time for quinine was approximately 200 s with a sensitivity enhancement factor of approximately 61 when compared to the typical gated injection. The detection and quantification limits of the developed approach for quinine were 3.0 μg/mL and 10 μg/mL, respectively, with intraday and interday repeatability (%RSDs, n = 5) of 3.6 and 4.4%. Recoveries in spiked human plasma were 98.1–99.8%.     

Open source simulation tool for electrophoretic stacking,focusing, and separation

We present the development, formulation, and performance of a new simulation tool for electrophoretic preconcentration and separation processes such as capillary electrophoresis, isotachophoresis, and field amplified sample stacking. The code solves the one-dimensional transient advection-diffusion equations for multiple multivalent weak electrolytes (including ampholytes) and includes a model for pressure-driven flow and Taylor–Aris dispersion. The code uses a new approach for the discretization of the equations, consisting of a high resolution compact scheme which is combined with an adaptive grid algorithm. We show that this combination allows for accurate resolution of sharp concentration gradients at high electric fields, while at the same time significantly reducing the computational time. We demonstrate smooth, stable, and accurate solutions at current densities as high as 5000 A/m² using only 300 grid points, and a 75-fold reduction in computational time compared with equivalent uniform grid techniques. The code is available as an open source for free at http://microfluidics.stanford.edu.     

Use of Counterflow in Bidirectional Capillary Isotachophoresis

Electrochemical cell using counterflow in bidirectional capillary isotachophoresis is designed. With this cell, steady-state concentrations of several solutions in cationic and anionic capillary isotachophoresis are determined simultaneously. Transport numbers of ionic components are calculated and compared with literature data.     

Automated free-solution isotachophoresis: Instrumentation and fractionation of human serum proteins

An automated free-solution isotachophoresis system (FS-ITP) for preparative fractionation of biopolymers is described, operated in a batch mode. The dimension of the separation chamber allows an up to 1200-fold higher sample load compared to separation in capillaries of 180 μm inner diameter as used in analytical capillary isotachophoresis (C-ITP). The preparative capacity of the system is within the milligram range. The method is fully compatible with analytical C-ITP, which is essential for preparative-scale isotachophoresis with regard to optimization of electrolyte systems and the search for suitable spacers. As a model application the fractionation of human serum proteins is reported. The collected fractions were analyzed by C-ITP and agarose gel electrophoresis.