Quantitation and on-line concentration of enantiomers in open-tubular capillary electrochromatography.

The feasibility of open-tubular capillary electrochromatography (OTCEC) with UV detector for quantitation of enantiomers is explored, and a simple on-line sample concentration method to improve detection sensitivity of negatively charged enantiomers more than 1000-fold is described. With a capillary of 25 microm ID, the limits of detection (LODs) for absolute concentration and for enantiomeric ratio are 10(-6) M and 0.6-0.8% (signal-to-noise ratio S/N = 10). Good linearity and reproducibility are observed. The detection sensitivity is enhanced by combination with field-enhanced sample injection (FESI). A water plug is introduced hydrodynamically into the capillary inlet end and then the sample solution prepared with water is introduced with electrokinetic injection. With this concentration technique, the LOD for absolute concentration is reduced to a 10(-9) M level. On the other hand, due to the peak-sharpening effect of FESI, the LOD for enantiomeric ratio for the first-eluted enantiomer is significantly improved, being 0.3%. Effects of the injection conditions, such as length of water plug, buffer concentration, injection voltage, and injection time on the enrichment efficiency are investigated. Online concentration of a racemic compound with two chiral centers is demonstrated.     

Principle and applications of the partial filling technique in capillary electrophoresis

Summary The partial filling technique (PFT) in capillary electrophoresis (CE) is an efficient system where, only 50–800 nanolitres of a chiral selector solution needs to be added to each run. PFT is especially applicable when these additives to the background electrolyte (BGE) are expensive or absorb UV light. The selector dissolved in the BGE is applied to the capillary as a plug, shorter than the effective length of the capillary, prior to application of the analyte. During the run both ends of the capillary are connected to the BGE. The applied plug and the analyte may move in opposite directions or in the same direction at different velocities depending on their electrophoretic mobilities. Thus the final plug length is either longer or shorter than the original length. The technique has been successfully applied in a number of studies including enantiomeric separation with a variety of selectors, and for the determination of conditional association constants. Taken from Dr. Pharm. Sc. Thesis, A. Amini, Uppsala, 1998.     

Capillary electrophoretic analysis of alkaline phosphatase inhibition by theophylline

An analytical method for studying enzyme inhibition has been developed using capillary electrophoresis with laser-induced fluorescence detection. This technique is based on electrophoretic mixing of zones of enzyme and inhibitor in substrate-filled capillaries. Enzyme catalytic activity is measured by detecting the fluorescent reaction product as it migrates past the detector. Reversible enzyme inhibition is indicated by a transient decrease in product formation. The enzyme, alkaline phosphatase, has been studied using the fluorogenic substrate AttoPhos ([2,2'-bibenzothiazol]-6-hydroxy-benzthiazole phosphate). This assay has been used to quantify theophylline, a noncompetitive, reversible inhibitor of alkaline phosphatase. The detection limit for theophylline is estimated at 3 microM, and 8.6 amole of alkaline phosphatase are required for each assay. The calculated K(i) for theophylline is 90 microM for the capillary electrophoretic enzyme-inhibitor assays.     

利用泰勒分析和动态涂层毛细管电泳系统快速测定蛋白质的扩散系数

以泰勒分散理论为基础, 首次采用动态涂层毛细管来准确和快速测定蛋白质分子的扩散系数.     

Analytical potential of enzyme-coated capillary reactors in capillary zone electrophoresis

Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples.     

Integrated 2-D CE

A simple methodology for converting a commercial CE-MS instrument into an integrated 2-D CE system has been developed. The first-dimensional capillary operates as a typical CE instrument with UV/visible detection. Fractions leaving the first dimension are automatically collected and introduced into the second dimension, performed on a CE-MS apparatus, for analysis. The integrated system allows fractions in the second dimension to be analyzed using various electrophoretic modes. As an example, in this work we performed the separation of two families of antibiotics (nitroimidazoles and tetracyclines) in the first dimension and the subsequent resolution of the antibiotics in each family (nitroimidazoles were resolved by MEKC and tetracyclines by CZE) in the second dimension. The proposed system, which operates in an highly automatic manner, is flexible and allows various combination of electrophoretic modes to be implemented. In addition, the use of a mass spectrometer detector in the second dimension further increases the analytical potential of the system as a result of the high selectivity and wealth of structural information provided by the MS detector.     

Reversed-polarity capillary zone electrophoretic analysis of usnic acid.

A capillary zone electrophoretic (CZE) method for the determination of usnic acid is described for the first time. Usnic acid is an antibiotic substance from lichens. Due to its low solubility in water, a high content of methanol in CZE buffer is required. Because of the methanol in the buffer, the electroosmotic flow velocity was lower than the electrophoretic mobility of usnic acid. Accordingly, the use of reversed-polarity (with the anode on the detector side of the capillary) was necessary. The optimal buffer composition was 50 mM NaOH, 20 mM acetic acid and 5% water in methanol. The detection limit of UV detector at 290 nm for usnic acid in the injected extract was 3.5 mg/L and the relative standard deviation of the normalized peak area was 3.3% at 250 mg/L.     

Immobilized capillary enzyme reactor based on layer-by-layer assembling acetylcholinesterase for inhibitor screening by CE

A method for creating an immobilized capillary acetylcholinesterase (AChE) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. The unique capillary AChE reactor was easily prepared by the instrument in three steps: first, a 0.5 cm long plug of a solution of the cationic polyelectrolyte polydiallyldimethylammonium (PDDA) was injected into the capillary to produce a positively charged coating on the surface of the capillary; subsequently, the enzyme solution with the same plug length was injected into the capillary and incubated for 10 min to immobilize the enzyme on the capillary wall via electrostatic interaction; third, PDDA solution with the same plug length was injected again into the capillary to cover the immobilized enzyme by forming PDDA-AChE-PDDA sandwich-like structure. The enzyme reactor can be easily renewed after removing the immobilized enzyme by flushing the column with 1 M NaCl solution. Activity of the immobilized enzyme can be assayed simply by carrying out an electrophoretic separation, i.e., the substrate solution was injected and incubated for a short time, followed by applying a voltage to separate the product from the unreacted substrate. The measured peak area of the product then represented the enzyme activity. For enzyme inhibitor screening, the mixture solution of the substrate and the inhibitor was injected and assayed the reduction of the enzyme activity. The immobilized enzyme could withstand 100 consecutive assays by only losing 10% activity. The reproducibility in terms of time-to-time, day-to-day, and batch-to-batch was measured with RSD% less than 4.7%. Furthermore, the screening system was validated by a known inhibitor. Finally, screening a small compound library containing four known AChE inhibitors and 42 natural extracts was demonstrated, and species with inhibition activity can be straightforwardly identified with the system.     

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

CE was used for the first time to study the two‐substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine‐5′‐triphosphate were in‐capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine‐5′‐triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero‐potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine‐5′‐diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose‐1,6‐bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in‐capillary mixed using an electrophoretically mediated microanalysis approach.     

Multi-compound electrophoretic assays for tyramine oxidase with a UV area detector imaging multiple windows on a looped capillary

Active pixel sensor UV area imaging and capacitively coupled contactless conductivity detection have been applied in an electrophoretically mediated microanalysis (EMMA) assay for substrate specificity of tyramine oxidase (Arthrobacter sp.). Use of the UV area imaging detector to monitor four windows in a capillary with three loops provided intrinsic self-referencing for all species and identified tyramine and 2-phenethylamine as the only reactive components in a multi-compound mixture. Continuous engagement EMMA experiments showed significant benefits by comparison with plug-plug EMMA, improving sensitivity by extending enzyme-substrate interaction times and allowing measurement of time-dependent reaction in the substrate zones passing the four windows.     

Determination of enzymatic activity and properties of secretory phospholipase A2 by capillary electrophoresis.

A capillary electrophoretic (CE) system coupled with a diode array UV detector was used for the assay of secretory phospholipase A2 (sPLA2) activity. This method is based on monitoring both the breakdown of substrates and the formation of products simultaneously using micellar electrokinetic chromatographic techniques. Under our developed separation conditions, we analyzed the substrates and products quantitatively, and investigated enzyme activity as a function of reaction time and presence of enzyme activator or inhibitor. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was also utilized to confirm the phosphatidylcholine, a substrate of sPLA2. In order to test the feasibility of the developed method for measurement of enzymatic activity, we compared it to the conventional radioactive assay method for sPLA2. On the basis of our results, the conventional method can be complemented, or even replaced, by this new CE method which possesses the advantages of short analysis time, use of non-radiolabeled and inexpensive substrates, simple measurement of enzymatic activity, and exact quantitation of substrate and product.     

Large-volume stacking in capillary electrophoresis using a methanol run buffer

Highly sensitive nonaqueous capillary electrophoresis of weakly acidic organic compounds has been performed using methanol as the run buffer solvent. Methanol provided appropriate suppression of the electroosmotic flow and an increase in the electrophoretic mobilities of anionic solutes compared to water. These two effects allowed large-volume stacking using the electroosmotic flow pump (LVSEP) to be achieved for larger anions using a bare fused-silica capillary under an electric field of reverse polarity, whereas only fast-moving small anions were previously known to be suitable for LVSEP in aqueous media. A field-enhanced sample injection of an additional amount of analytes during the solvent plug removal further enhanced the limits of detection to below the nanomolar range with conventional UV absorption detection. Under optimum conditions, excellent linear responses and reproducibility in the migration times together with the corrected peak areas for ten analytes were obtained in the concentration range of 10-100 nM.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Evaluation of quail egg white riboflavin binding protein as a chiral selector in capillary electrophoresis by applying a modified partial filling technique.

A preliminary evaluation of the enantioselective properties of quail egg yolk riboflavin binding protein (qRfBP) was carried out in capillary electrophoresis by using the complete filling technique. The most promising results obtained by this screening of nineteen chiral drugs were singled out with the aim of optimizing enantiomer separations by applying the partial filling technique, which allows operating at much higher protein concentrations without detection problems. The building of the separation zone in the partial filling technique has been modified in order to enable on-line monitoring, before each run, of the actual protein plug application velocity and, consequently, the building of a plug of the desired length. The electrophoretic conditions chosen gave opposite migration directions for the chiral selector and the analytes, with qRfBP migrating away from the detector. A polyvinyl alcohol-coated capillary was first totally filled with protein and the optimal plug length was obtained by further applying negative pressure together with positive voltage for the time needed. Separations of basic drugs were optimized by using protein concentrations ranging from 200 microM up to 900 microM and different plug lengths, while the running buffer pH (6.0), temperature (25 degrees C) and operating voltage (+20 kV) were kept constant. The enantioresolution of all solutes was affected by both the chiral selector concentration and protein plug length. Baseline separations were obtained for oxprenolol, prilocaine and bupivacaine.     

Simultaneous microchip enzymatic measurements of blood lactate and glucose

A miniaturized capillary electrophoretic (CE) microchip device for the simultaneous measurements of lactate and glucose is described. The new microchip bioassay protocol integrates an electrophoretic separation of lactate and glucose, post-column enzymatic reactions of these metabolites with their respective oxidase enzymes, and an amperometric (anodic) detection of enzymatically-liberated hydrogen peroxide at a gold-coated thick-film carbon detector. Factors influencing the response have been examined and optimized, and the analytical performance has been characterized. Applicability of the microchip assay to clinical samples, such as serum and blood, is demonstrated. The microchip protocol obviates cross enzymatic reactions and interferences from major oxidizable constituents common to dual glucose-lactate enzyme electrodes. Such ability to rapidly separate and quantitate lactate and glucose on a small microchip platform should find important clinical and biotechnological applications.     

Predefine resolution of enantiomers in partial filling capillary electrophoresis and two discontinuous function plugs coupling in‐capillary

In common partial filling CE (PF‐CE), the capillary contains the selectors plug between the injection and detector end to avoid the selector going into the detector zone. To expand this method, we propose a mode of two discontinuous function plugs coupling in‐capillary, named as plug–plug PF‐CE (ppPF‐CE). Initially, we present the method to predefine the effective length of chiral selector to meet the requirement of enantiomers' resolution, which could avoid some experimental procedures. With α‐CD as a chiral selector, a satisfactory resolution of enantiomers d,l ‐tryptophan and d,l ‐tyrosine was obtained with a partial filling α‐CD plug of optimal length and concentration. Subsequently, a second plug containing hydroxypropyl methylcellulose, organic solvents (acetonitrile and methanol), anionic and cationic surfactants (SDS and CTAB), and different concentrations of sodium phosphate buffer was inserted after the selector plug. Effects of plug–plug filling on enantiomers' migration and resolution are discussed. The ppPF‐CE might be a new flexible mode for CE application.     

Design and optimization of on-chip capillary electrophoresis

We present a systematic, experimentally validated method of designing electrokinetic injections for on-chip capillary electrophoresis applications. This method can be used to predict point-wise and charge-coupled device (CCD)-imaged electropherograms using estimates of species mobilities, diffusivities and initial sample plug parameters. A simple Taylor dispersion model is used to characterize electrophoretic separations in terms of resolution and signal-to-noise ratio (SNR). Detection convolutions using Gaussian and Boxcar detector response functions are used to relate optimal conditions for resolution and signal as a function of relevant system parameters including electroosmotic mobility, sample injection length, detector length scale, and the length-to-detector. Analytical solutions show a tradeoff between signal-to-noise ratio and resolution with respect to dimensionless injection width and length to the detector. In contrast, there is no tradeoff with respect to the Peclet number as increases in Peclet number favor both SNR and separation solution (R). We validate our model with quantitative epifluorescence visualizations of electrophoretic separation experiments in a simple cross channel microchip. For the pure advection regime of dispersion, we use numerical simulations of the transient convective diffusion processes associated with electrokinetics together with an optimization algorithm to design a voltage control scheme which produces an injection plug that has minimal advective dispersion. We also validate this optimal injection scheme using fluorescence visualizations. These validations show that optimized voltage scheme produces injections with a standard deviation less than one-fifth of the width of the microchannel.     

Electrophoretically mediated microanalysis of a nicotinamide adenine dinucleotide-dependent enzyme and its facile multiplexing using an active pixel sensor UV detector

An electrophoretically mediated microanalysis (EMMA) method has been developed for yeast alcohol dehydrogenase and quantification of reactant and product cofactors, NAD and NADH. The enzyme substrate ethanol (1% (v/v)) was added to the buffer (50 mM borate, pH 8.8). Results are presented for parallel capillary electrophoresis with a novel miniature UV area detector, with an active pixel sensor imaging an array of two or six parallel capillaries connected via a manifold to a single output capillary in a commercial CE instrument, allowing conversions with five different yeast alcohol dehydrogenase concentrations to be quantified in a single experiment.     

Rapid analysis of atorvastatin calcium using capillary electrophoresis and microchip electrophoresis

In this work, a capillary electrophoretic method for the rapid quantitation of atorvastatin (AT) in a lipitor tablet was investigated and developed. Method development included studies of the effect of applied potential, buffer concentration, buffer pH, and hydrodynamic injection time on the electrophoretic separation. The method was validated with regard to linearity, precision, specificity, LOD, and LOQ. The optimum electrophoretic separation conditions were 25 mM sodium acetate buffer at pH 6, with a separation voltage of 25 kV using a 50 microm capillary of 33 cm total length. Sodium diclofenac was used as an internal standard. Analysis of AT in a commercial lipitor tablet by electrophoresis gave quite high efficiency, coupled with an analysis time of less than 1.2 min in comparison to LC. Once the separation was optimized on capillary, it was further miniaturized to a microchip platform, with linear imaging UV detection using microchip electrophoresis (MCE). Linear imaging UV detection allowed for real-time monitoring of the analyte movement on chip, so that the optimum separation time could be easily determined. This microchip electrophoretic method was compared to the CE method with regard to speed, efficiency, precision, and LOD. This work represents the most rapid and first reported analysis of AT using MCE.     

Development of a microbioreactor with ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) immobilized on a polyacrylamide-coated capillary at the outlet

A simple and fast method of immobilization of cell membrane suspension containing human ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) on a polyacrylamide-coated capillary was developed. The enzyme microbioreactor was prepared by hydrodynamic injection of a small plug of the polycationic electrolyte hexadimethrine bromide (HDB) followed by a suspension of an enzyme-containing membrane preparation. In order to shorten the enzyme assay time and to increase the throughput of the assay, the capillary was coated from the outlet end and all injections were performed from the outlet end of the capillary. For the monitoring of the enzymatic reaction, the substrate ATP dissolved in reaction buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM Hepes, pH 7.4, internal standard: 10 μM UMP) in the absence or presence of inhibitor was injected electrokinetically and incubated in the microbioreactor for 1 min with 1 kV of applied voltage. Then, the electrophoretic separation of the reaction products was initiated by applying a constant current of 60 μA. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by UV absorbance at 260 nm. The new method was compared with an at-capillary-inlet method without immobilization of the enzyme. The results (K_m values, K_i values for inhibitor) obtained with both methods were similar and comparable with literature data. The developed outlet immobilized enzyme microreactor using a coated capillary is very fast, simple and most economic allowing multiple use of the enzyme.