Flavonoid separation by capillary electrophoresis. Effect of temperature and pH

Summary The use of capillary electrophoresis for the analysis of selected flavonols present in fruit juices and wines (kaempferol-3- rutinoside, rutin, avicularin, quercitrin, isoquercitrin, isorhamnetin, kaempferol and quercetin) was explored, and the effect of pH and temperature on the separation studied. The method had good reproducibility and analyses were carried out in less than 10 minutes.     

pH programming in capillary electrophoresis by means of temperature programming

When using a background electrolyte with a buffer having strong temperature dependence of pK, different operating temperatures result in different operating pH values, using the same background electrolyte (BGE). It has been shown that this can be used to fine-tune selectivities of sample mixtures of weak analytes. Capillary electrophoresis (CE) equipment is designed to operate under isothermal conditions, but by proper programming, a very reproducible temperature and thus BGE pH program during the analysis can be realized. This was experimentally verified and illustrated by computer simulations. Equipment characteristics have been determined, and possibilities and restrictions to make use of this feature are presented.     

Influence of temperature control in capillary electrophoresis

We have investigated the influence of capillary temperature on migration time and peak area and have evaluated different cooling systems. It was found that for applied voltages below 15 kV (i.e. those most frequently used) temperature control effectively improves peak area reproducibility but has less effect on migration time.     

Temperature effects in capillary electrophoresis. 1: Internal capillary temperature and effect upon performance

Summary In isothermal CE the migration velocity of analytes and the number of theoretical plates delivered are expected to be proportional to the field strength. In reality ohmic heating of the capillary causes distortions: the migration velocity increases more rapidly while the plate count increases less rapidly, and may even fall at high values of the field. These distortions are worse the larger the bore of the capillary and the higher the concentration of buffer. A detailed investigation of these effects using capillaries cooled by natural convection has confirmed that self heating of the capillary is indeed largely responsible. The extent of self heating has been determined by three independent methods and to a first approximation is proportional to the power dissipation in the capillary. Decreasing viscosity with temperature is responsible for the nonlinearity of the dependence of velocity upon field strength while increase in the diffusion coefficient of analytes is responsible for the poorer than expected performance at high field strengths.     

Prediction of the separation of phenols by capillary zone electrophoresis

The effect of pH and ionic strength on the migration of neutral acids in capillary zone electrophoresis (CZE) has been studied for several phenols. The mobilities of the phenols and the efficiency of the capillary have been related to the studied factors. The mobility can be related to the pH of the running buffer through the mobility of the phenolate ion, and the conditional acidity pK value of the phenol at the working ionic strength. This allows prediction of the migration of the phenol, solely from its pK_a^′ value (literature pK_a corrected for the ionic strength of the solution) and mobility of the anion, which can be easily calculated from the mobility at a basic pH value and the pK_a^′ value. Combination of the predicted mobility with the efficiency allows estimation of the resolution of the consecutive peaks obtained for a mixture of phenols. This method has been tested for two groups of phenols of environmental interest.     

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.     

Optimisation of the separation of the dimethylpyridines by capillary electrophoresis

The separation of the isomers of dimethylpyridine has been studied by capillary electrophoresis. Studies at low pH where the dimethylpyridines all bear the same charge showed a partial separation probably due to size/shape effects. The charge profiles of the analytes calculated from their ionisation constants were compared with their separation between pH 2.5 and 9. The optimum resolution was found at a pH of 6.5 in the area of maximum charge difference between the dimethylpyridines.     

Infrared-based temperature measurements in capillary electrophoresis

In capillary electrophoresis (CE), the temperature inside the capillary is one of the most important parameters. In a concept for Analytical Instrument Qualification (AIQ) of CE systems, the temperature accuracy and stability have to be included. This fact requires an accurate look at the measurement of temperature which is generated by the applied electrical power. The generation of Joule heating is measured on the outside of the capillary using an infrared (IR) thermometer. The thermometer linearity is demonstrated over a wide range of various electrical field strength, buffer systems, and different capillary inner diameters. A slope of 6.3 °C m/W was found for the optimal thermometer capillary distance of 8 mm. Furthermore, the temperature measurements are highly precise, depending almost solely on the current variability. The proposed method is compared with three methods calculating the temperature from the conductance, the electroosmotic velocity, or the current. These indirect methods estimate slopes ranging from 7 to 10 °C m/W. In addition, the maximal suitable electrical power per unit length is estimated. Joule heating can often be tolerated up to 4 W/m. However, sensitive analytes can already be affected by using more than 1 W/m. In conclusion, the consideration of the temperature is essential for not only Analytical Instrument Qualification, but also certainly useful for method optimisation and method transfer.     

Thermostating in capillary electrophoresis

Summary The use of high voltages across a electrophoresis capillary will increase the temperature of the buffer due to Joule heating. As a result temperature control in CE is rather important since variations in the buffer temperature will result in changes in the pH of the buffer, peak shape, migration time, reproducibility, efficiency, 3-D structure of macromolecular analytes, etc. Six different thermostating systems have been evaluated: (i) natural convection, (ii) fan, (iii) home-made and (iv and v) two commercially available high-speed air and a (vi) liquid thermostated device. In all cases the temperature of the buffer in the capillary is calculated according to the temperature-conductivity relationship. For this purpose two parameters are introduced describing temperature control: the temperature onset (δT) and the temperature rise factor (α). From these results, it can be concluded that high speed air thermostating can be as efficient as liquid thermostating.     

Characterization of single-stranded DNA separation by capillary gel electrophoresis

We have investigated the effect of polymer gel reconditioning, the shape of the capillary, the applied electric field, and the capillary length for single-stranded DNA. The polyethylene oxide gel had deformed under the high electric field causing the degradation of the separation power. By the reintroduction of the fresh polyethylene oxide gel for the next run, one-base resolution was recovered. It turned out that the tip of the capillary at the injection side needed to be clean and symmetric for much improved resolution. Changing DNA motion by the pulsed electric field resulted in the separation of DNA far more than 500 bases.     

Carbon nanotubes as separation carrier in capillary electrophoresis

The utility and versatility of carboxylic single-walled carbon nanotubes (c-SWNT) in capillary electrophoresis (CE) is demonstrated, using as model solutes homologues and structural isomers. In the case of homologues of caffeine and theobromine, distinct changes in the electrophoretic parameters occur at a critical concentration of c-SWNT in the run buffer. It is suggested that the c-SWNT of a definite concentration could form a network in the run buffer as a pseudostationary phase on the basis of the unique tubule structure, providing a different separation from sodium dodecyl sulfate (SDS) micelles. In the case of structural isomers of catechol and hydroquinone, differing from the homologues, it is mainly attributable to the functional groups on the c-SWNT that have an effect on the electrophoretic behaviors by forming intermolecular hydrogen bonding with analytes. Furthermore, aggregated c-SWNT serve as anticonvective media and minimize solute diffusion contributing to zone broadening. The presence of charged c-SWNT suppressed the electrodiffusion and decreased the adsorption between capillary wall and solutes, which led to better peak shapes of isomers.     

Isotopic separation of lithium ions by capillary zone electrophoresis

Separation of ⁶Li and ⁷Li isotopes by CZE was demonstrated. The BGE contained 5 mM 4‐aminopyridine, 0.9 mM oxalic acid, 0.25 mM CTAB, and 0.25% w/v Tween 20 (рН = 9.2). The running conditions were +25 kV at 30°C with indirect photometric detection at 261 nm. Under optimal experimental conditions, the analysis time was less than 21 min. Separation of Li preparations with mole fraction of ⁶Li ranging from 3.44 up to 90.38% was demonstrated.     

Simultaneous separation of nitrate, nitrite and ammonium by capillary electrophoresis

Summary A capillary electrophoretic method for the simultaneous separation of nitrate, nitrite and ammonium has been developed. Direct (NO₃ ⁻, NO₂ ⁻) and indirect (NH₄ ⁺) UV detection at 214 nm in conjunction with electromigration sampling from both ends of the capillary was used. Two electrolyte systems based on imidazole-sulfate (pH 3.8) and copper(II)-ethylenediamine-chloride (pH 8.0) were investigated. Optimisation of the experimental parameters such as electrolyte concentration, pH, nature of the counter-ion, was studied. The method permits excellent separation of three nitrogen species in only 4 min. The analytical performance of both electrolyte systems is compared in terms of migration time and peak area repeatability and detectability. Alkaline electrolyte shows a better overall analytical performance.     

毛细管区带电泳法拆分手性药物环扁桃酯

近年来,随着不同种类的手性添加剂[1]在毛细管电泳(CZE)中的使用,毛细管电泳越来越显示出其强有力的手性拆分性能。具有特殊笼状结构并含有多个手性中心的环糊精及其衍生物是毛细电泳手性分离研究中最常采用的手性添加添[2-4]。本文合成了环糊精衍生物单3 O 苯基胺甲酰基 β CD[2]并以之作为手性选择剂分离了β CD及手性药物环扁桃酯。1　实验部分932 3 HVPS高压电源(山东省化工研究院),DD 2000型可调波长紫外检测器(中国科学院大连化学物理研究所),XWT型记录仪(上海大华仪表厂),pHS 25型酸度计(上海雷磁仪器厂),石英毛细管45cm…     

毛细管电泳分离几种神经递质的研究

研究了不同毛细管预处理方法对组胺、5-羟色胺及儿茶酚胺类神经递质电泳分离的影响，采用优化的毛细管预处理方法及电泳分离条件基线分离了组胺、5-羟色胺、去甲肾上腺素和肾上腺素。利用多巴胺和5-羟色胺在毛细管内壁吸附效应的不同，对电泳淌度极为相近的多巴胺和5-羟色胺进行了分离和鉴定。以PC22细胞为研究对象，证实了该细胞中所含大量神经递质是多巴胺，不是5-羟色胺。     

Rapid separation of acetophenone and its monohydroxy isomers by capillary electrophoresis

This work describes the development of a capillary electrophoresis(CE)method for the simultaneous separation of acetophenone(AP),2-hydroxyacetophenone(2-HAP),3-hydroxyacetophenone(3-HAP)and 4-hydroxyacetophenone(4-HAP)in synthetic mixtures using 10 mmol/L of sodium tetraborate buffer(pH 9.5).The aim of this work is to demonstrate the effectiveness of CE to separate AP and its monohydroxy isomers and to defne how the separations are affected by buffers,buffer pH,sample matrices and separation voltage.This method was successfully used for the trace level separation and determination of 2-HAP,3-HAP and 4-HAP in synthetic mixture and 4-HAP in spiked plasma samples.     

Enantioselective separation of rivastigmine by capillary electrophoresis with cyclodextrines

Different beta-cyclodextrines (beta-cyclodextrin, heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and sulfated beta-cyclodextrin) were investigated as additives for the enantioselective separation of the R-form from rivastigmine ((S)-N-ethyl-3-[(1-dimethylamino) ethyl]-N-methyl-phenyl carbamate), contained as impurity in this drug, which is used for the treatment of Alzheimer's disease. Electrophoresis was performed in an acidic background electrolyte (triethanolammonium phosphate, 75 mM, pH 2.5) with various concentrations of the additives. The electrophoretic mobilities measured are typical functions of the additive concentrations, with complex constants (obtained by fitting the appropriate binding curve on the data) ranging between about 180 and 770 M(-1). Best separation was obtained with 7.5 mM beta-cyclodextrin, with the R-enantiomer as impurity migrating before the main S-compound. Intra- and interday reproducibility (n = 6 and 18, respectively) of migration time and peak area was in the low percentage range, linearity of the calibration line for the quantitation of the impurity in the range between 2.3 and 50 microg/ml, expressed by the linear correlation coefficient, was 0.9998. The limits of detection and quantitation, respectively, were 0.7 and 2.3 microg/ml, corresponding to 0.05 and 0.15%, m/m of the R- relative to the S-compound. Analysis can be carried out at 18 degrees C in less than 19 min.     

Variation of the pH of the background electrolyte due to electrode reactions in capillary electrophoresis: Theoretical approach and in situ measurement

Electrode reactions during the electrophoretic process may change the pH of the buffer and subsequently the migration behavior of solutes with resultant loss of reproducibility. A theoretical treatment of pH variations due to electrolytic processes is presented. The choice of buffer appears to have a dramatic influence on the pH variations observed, even if substantial buffer action is expected at the pH chosen. The experimental evaluation of the separation of 4-hydroxy-3-methoxycinnamic acid and 3-hydroxybenzoic acid reveals that the quality of the separation decreases continuously from a baseline separation observed in the first experiment to a comigration of the two solutes (resolution = 0) in the ninth experiment. A pH decrease of about 0.05 pH units accounts for the observed changes in mobility. A novel in situ pH measurement approach is presented, in which the mobility, peak area, and peak height of an indicator dye are related to the pH in the capillary. This enables the identification and quantification of pH variations during electrophoretic runs: the pH decreases at the anodic side already after the first experiment and pH variations as small as 0.02 pH units can be measured. The variations in peak height appear to be less suited. The calculated pH variations are in close agreement with the ones obtained experimentally.