Advances in capillary electrophoretically mediated microanalysis

Recent developments in the use of capillary electrophoretic techniques for the in-line study of enzyme reactions and derivatization protocols are reviewed. The article is divided into two parts: (i) in-line enzyme reactions and (ii) in-line derivatization. The first part introduces electrophoretically mediated microanalysis (EMMA) and discusses and illustrates the different modes of EMMA. A literature overview is provided, starting from 1996, and the investigated enzymes are classified into two tables based on the mode of engagement (i.e., continuous or transient) of the developed EMMA-based assay. The second part starts with an introduction of the procedures and the nomenclature used in the area of in-line derivatization protocols based on EMMA. Reported derivatization procedures are discussed and classified in tables, according to the functional group that is derivatized.     

Advances in CE-mediated microanalysis: an update

This review, as a continuation of two earlier reports, gives an overview of the recent developments, over the period from 2005 until now, in the use of electrophoretically mediated microanalysis (EMMA) methodology for the on-line study of enzymatic reaction and derivatization. The article is divided into two parts: (i) on-line enzymatic reaction by EMMA and (ii) on-line derivatization by EMMA. Following a brief introduction, a literature overview on enzymatic reaction is provided. The second part starts with an introduction of the purpose of derivatization and the nomenclature used in the area of in-capillary derivatization based on EMMA. The development of more integrated analytical platform that combines in-capillary derivatization and sample preconcentration is discussed. Reported derivatization procedures are summarized.     

Recent developments and applications of EMMA in enzymatic and derivatization reactions

This review provides systematic coverage of examples in the field of in-capillary electrophorecially mediated microanalysis (EMMA). The recent developments and applications in the time period up to mid 2011 have been described, as well as relevant older papers. The basic principles and modes of in-capillary assays have been demonstrated. An overview is also given of the various injection, separation and detection modes implemented in combination with EMMA. The review is presented in two parts mainly dealing with (i) enzymatic and (ii) derivatization or chemical reactions. Finally, the future trends of CE in performing and monitoring reactions have been drawn.     

Investigating the effects of conductivity on zone overlap with EMMA: computer simulation and experiment

In this paper, we demonstrate, using both experiment and simulation, how sample zone conductivity can affect plug-plug mixing in small molecule applications of electrophoretically mediated microanalysis (EMMA). The effectiveness of in-line mixing, which is driven by potential, can vary widely with experimental conditions. Using two small molecule systems, the effects of local conductivity differences between analyte plugs, reagent plugs and the BGE on EMMA analyses are examined. Simul 5.0, a dynamic simulation program for CE systems, is used to understand the ionic boundaries and profiles that give rise to the experimentally obtained data for EMMA analyses for (i) creatinine determination via the Jaffe reaction, a reaction involving a neutral and an anion, and (ii) the redox reaction between gallate and 2,6-dichloroindophenol, two anions. Low sample conductivity, which is widely used in CE analyses, can be detrimental for in-line reactions involving a neutral reactant, as rapid migration of the ionic component across a low conductivity neutral zone results in poor reagent plug overlap and low reaction efficiency. Conversely, with two similarly charged reagents, a low conductivity sample plug is advantageous, as it allows field-amplified stacking of the reagents into a tight reaction zone. In addition, the complexity of simultaneously overlapping three reagent zones is considered, and experimental results validate the predictions made by the simulation. The simulations, however, do not appear to predict all of the observed experimental behavior. Overall, by combining experiment with simulation, an enhanced appreciation for the local field effects in EMMA is realized, and general guidelines for an advantageous sample matrix can be established for categories of EMMA analyses.     

Off-line and in-line determination of catechol- O-methyltransferase activity in a capillary electrophoretic system

The use of capillary electrophoresis for the determination of catechol-O-methyltransferase (COMT) activity with dihydroxybenzoic acid as a substrate was investigated. Both an off-line and in-line capillary electrophoresis determination of COMT activity was developed and the two approaches are discussed. In the presented methods, substrate and reaction products are monitored at the same time. The initial velocity of the reaction is quantified spectrophotometrically by the corrected peak area of the products at 200 nm. In the off-line setup, capillary zone electrophoresis is used to separate and quantify the different reaction compounds. Each electrophoretic run required only 37 nL of the enzymatic reaction solution. Based on the off-line assay, an in-line determination of COMT activity was developed by a methodology known as electrophoretically mediated microanalysis (EMMA). All the different steps (i.e. mixing, incubation, separation and in-line quantitation) are combined in the capillary, which is used as a microreactor for the enzymatic reaction. Full automation of the assay is achieved with this microscale approach.     

Electrophoretically mediated microanalysis

This review describes the existing developments in the use of the capillary electrophoretic microanalytical technique for the in-line study of enzyme reaction, electrophoretically mediated microanalysis (EMMA). The article is divided into a number of parts. After an introduction, the different modes, basic principle, procedure, and some mathematical treatments of EMMA methodology are discussed and illustrated. The applications of EMMA for enzyme assay and for non-enzymatic determination are summarized into two tables. In addition to classical capillary electrophoresis (CE) instrument EMMA, special emphasis is given to a relatively new technique: EMMA on CE microchip. Finally, conclusions are drawn.     

Electrophoretically mediated microanalysis of gentamicin with in-capillary derivatization and UV detection.

This paper describes a system for integration of a one-step-microscale chemical derivatization and analysis by a methodology known as electrophoretically mediated microanalysis (EMMA). Differential electrophoretic mobility between an analyte, reagent, and their product offers EMMA a unique capability to selectively carry out electrophoretic mixing, control product formation, and separation. This system was successfully applied to perform derivatization and separation of the multicomponent aminoglycoside antibiotic gentamicin using 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid as labeling reagents. A multivariate approach based on central composite experimental design was used to optimize the derivative yield. Full automation of the derivatization and analytical procedure, high derivatization efficiency, high sample throughput, and precision are the excellent features of the present method. In addition, this methodology offers short analysis time, as well as selectivity and sensitivity suitable for impurities determination. Separation of gentamicin C1, C1a, C2, C2a, C2b, sisomicin, and several minor components was achieved. For the first time separation and identification of three impurities, namely garamine, 2-deoxystreptamine, and paromamine are described.     

Derivatization in mass spectrometry --7. On-line derivatisation/degradation

The review describes on-line derivatization/degradation methods employed in mass spectrometry to solve some structural and analytical problems. Advantages and applications of various positions of reaction systems connected mainly to a mass spectrometer or a gas chromatograph/mass spectrometer are considered. Among these are reaction systems connected directly to the mass spectrometer (reaction mass spectrometry, pyrolysis-mass spectrometry or direct pyrolysis-mass spectrometry); flash-heaters as reactors in gas chromatography/mass spectrometry (GC/MS); in-line chemical reactors located before the chromatographic column [pre-column derivatization/degradation with the use of catalytic reactions, pyrolysis (pyrolysis-GC/MS), degradation in elemental analyzers-isotope ratio mass pectrometry (EA-IRMS)]; on-column derivatization and deuteration; reactor located between the chromatographic column and a mass spectrometer [post-column catalytic derivatization, gas chromatograph-combustion-isotope ratio mass spectrometer (GC-c-IRMS)]. Post-column derivatization in high performance liquid chromatography/mass spectro-metry is briefly mentioned. Application of such on-line methodology to structure elucidation of low molecular mass compounds and polymers, to the determination of isotope ratios of the most common elements, to the investigation of catalytic reactions is discussed..     

In-capillary determination of creatinine with electrophoretically mediated microanalysis: Characterization of the effects of reagent zone and buffer conditions

Previous work has demonstrated proof-of-concept for carrying out the clinically useful Jaffe reaction between creatinine and picrate within a capillary tube using electrophoretically mediated microanalysis (EMMA). Here, it is shown that careful control of reagent plug length as well as concentration and pH of the background electrolyte (BGE) can result in a marked improvement in the sensitivity of this assay. Increasing the length of the picrate reagent zone is shown to give rise to as much as a 3–4-fold enhancement, and increasing the concentration and/or pH of the borate buffer also results in an additional, albeit modest, improvement in sensitivity. Interestingly, borate BGE concentrations approaching 100 mM give rise to an unexplained drop in reaction efficiency, an effect which can be avoided by utilizing lower borate concentration with higher pH. The improvements appear to primarily minimize electrodispersion of the picrate reagent, allowing higher picrate concentration in the reaction zone. The same conditions also appear to minimize the electrodispersion of the in-line product as well. With optimized EMMA parameters, the sensitivity of the in-line Jaffe chemistry can be enhanced to an extent that there is no need for the two capillary “high sensitivity” detection system required in previous work. Using optimized conditions, three different human serum samples spanning the expected clinical range of creatinine concentrations were successfully analyzed. Overall, this work illustrates the importance of systematically characterizing the conditions under which EMMA analyses are carried out.     

Flow injection analysis coupled with HPLC and CE for monitoring chemical production processes

Summary In chemical and pharmaceutical production plants process control is often performed by plant personal near the process (at-line). Alternatively, spectroscopic procedures like near infrared may be coupled to the process using in-line or on-line interfaces. When the analytical problem cannot be solved by these established approaches chromatographic techniques can be directly coupled to the production process. An application of chemical reaction monitoring which is difficult to perform is reported. The analytical problems are solved by HPLC and CE. Both techniques in the application presented need an on-line derivatization step prior to the analysis. Flow Injection Analysis (FIA) was applied for this sample preparation step. For the online coupling to the commercially available CE-instrument a special sample vial was developed. The application shows sufficient reproducibility and analysis speed for near real-time monitoring. of the process. Dedicated to Professor Dr. Heinz Engelhardt on the occasion of his 65^th birthday.     

Determination of the kinetic parameters of rhodanese by electrophoretically mediated microanalysis in a partially filled capillary

Electrophoretically mediated microanalysis (EMMA) was applied for the study of kinetic parameters of the bisubstrate enzymatic reaction of rhodanese. The Michaelis constants (K(m)) for both substrates and the effect of temperature on rhodanese reaction were evaluated by means of the combination of the EMMA methodology with a partial filling technique. In this setup, the part of the capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. The enzymatic reaction was performed in 25 mM N-(2-hydroxymethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer (pH 8.5) while the low pH background electrolyte 100 mM beta-alanine-HCl (pH 3.5) was used for separation of substrates and products that are the inorganic anions. The estimated value of K(m) for thiosulfate of 1.30 x 10(-2) M was consistent with previously published values; the K(m) for cyanide of 7.6 x 10(-3) M was determined for the first time. In addition, the type of kinetic mechanism of enzymatic reaction was also elucidated. The finding of the double displacement (ping-pong) mechanism is in accordance with previous literature data. Also, the experimentally determined temperature optimum of the rhodanese-catalyzed reaction around 20-25 degrees C agreed with literature values.     

Functionalized carbon electrodes for pH determination

The derivatization of carbon materials has allowed electrochemists to develop electrodes for pH measurements that offer distinct advantages compared to other common methods for determining pH in solutions. In this report, an overview on recent work on the different derivatization strategies leading to potentiometric, amperometric, or voltammetric pH sensors is given. The topic is subdivided into five main categories: (1) chemical modification, (2) covalent bonding, (3) physical adsorption, (4) film formation, and (5) composite electrodes.     

Flow-Injection Analysis of Pharmaceuticals

The current state and outlooks of the development of the flow-injection analysis of medicinal substances in pharmaceuticals and biological fluids are considered. The role of chemical, photochemical and enzymatic reactions of derivatization in the flow-injection determination of pharmaceuticals is outlined. The role of detection methods in improving the selectivity and sensitivity of the flow-injection analysis of pharmaceuticals and expanding its possibilities in pharmaceutical analysis is considered     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。     

Electrophoretically mediated reaction of glycosidases at a nanoliter scale

We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of a native glyco-enzyme. As a representative of this class of enzyme, beta-glucosidase was selected, and the reaction was analyzed. Our EMMA was based on the plug-plug interaction of enzyme and substrate plugs, which is essential to reduce quantities of materials. Furthermore, we have addressed the problem of incompatibility of the enzymatic reaction and separation of the reactants. As a result, EMMA of native glycosidase was achieved with a reaction volume of approximately 20 nL and the Michaelis constant was estimated according to the Lineweaver-Burk plot. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme (ng order) and substrate (pmol order) required for a reaction.     

Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary

Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine-hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate--1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (K(M)) as well as the substrate inhibition constant (K(SI)). The value of K(M) and K(SI) obtained were 7.7+/-2.5 mM and 1.1+/-0.4 mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.     

Determination of Surface Functional Groups in Lignocellulosic Materials by Chemical Derivatization and ESCA Analysis

Simple and convenient methods for determining surface chemical composition of lignocellulosic materials are described. The methods are based on vapor phase fluorine surface derivatization with either trifluoro acetic anhydride (TFAA), tri-fluoro ethanol (TFE) or pentafluorophenyl hydrazine (PFPH) and subsequent Electron Spectroscopy for Chemical Analysis (ESCA). Model cellulosic surfaces with well defined functionalities were used to optimize the derivatization reaction conditions. Detection and accessibility of surface hydroxyl functional groups were investigated in cotton and regenerated cellulose as models. Carboxymethyl cellulose (CMC) was used as a model surface for detection and quantification of carboxylic acid groups. Theoretical conversion curves for derivatization reactions were calculated and used to evaluate the extent of the reactions on the model surfaces. It was found that the conversion was higher for the regenerated cellulose and CMC than for cotton. The protocols developed using the model surfaces were applied to a case study on wood fibers with different degrees of complexity, namely dissolving and chemithermomechanical (CTMP) pulp. Untreated and oxygen-plasma modified pulps were compared with respect to the surface composition of functional groups. According to the derivatization reactions, functionalities containing oxygen were significantly increased on the plasma-treated samples. The effect of the treatment was found to be dependent on the type of pulp. Fluorine derivatization is shown to be an unambiguous method for clear assessment of the chemical functionalities of cellulosic surfaces.     

Derivatization of inorganic ions in capillary electrophoresis

This review gives a short overview of the main approaches to the derivatization of inorganic ions in capillary electrophoresis (CE) with emphasis on the most recent works. Various derivatization procedures and detection methods are discussed. A brief account of their advantages and limitations is given. More specific areas such as microchip CE, simultaneous separation of anions and cations, and speciation analysis are also briefly discussed.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Characterization of Ethylene methyl methacrylate and Ethylene butylacrylate Copolymers with Interactive Liquid Chromatography

Summary: Copolymers of ethylene with methyl methacrylate (EMMA) and butyl acrylate (EBA), which are of different average chemical composition and block lengths according to NMR analysis, were analyzed by size exclusion chromatography (SEC), differential scanning calorimetry (DSC), Crystallization Analysis Fractionation (CRYSTAF), and high performance liquid chromatography at high temperature (HT-HPLC). With CRYSTAF and DSC crystallizing fractions were detected only in some samples. HT-HPLC fractionated all the samples irrespective of their crystallinity. Homopolymers, PMMA and PE were also found in the copolymer samples of EMMA. EMMA and EBA were separated in HPLC according to the content of polar comonomer. A linear correlation between the MMA content and elution volume could not be established due to the presence of homopolymers as admixtures. In such a case the average chemical composition obtained by NMR does not correspond to the real chemical composition of the copolymers. Unlike EMMA the EBA samples eluted in single peaks, which was used for evaluation of their chemical composition distribution. The comparison of results obtained by fractionation via CRYSTAF and HT-HPLC clearly demonstrates the advantages of the chromatographic approach to study the chemical heterogeneity of olefin based copolymers.