Inhibition Assay of Theophylline by Capillary Electrophoresis/Dynamic Frontal Analysis on the Hydrolysis of p-Nitrophenyl Phosphate with Alkaline Phosphatase

A novel inhibition assay is proposed by capillary electrophoresis/dynamic frontal analysis (CE/DFA). When a substrate of p-nitrophenyl phosphate and an inhibitor of theophylline were tandemly introduced into the capillary containing alkaline phosphatase as an enzyme, two plateau signals were detected in the electropherogram. A higher plateau is based on the CE/DFA without inhibition, and a suppressed plateau is formed under the inhibition while the substrate zone passing through the inhibitor zone. Inhibition constant was successfully determined through the two plateau heights

Determination of Two-Steps Acid Dissociation Constants of L-Ascorbic Acid by Capillary Zone Electrophoresis

Two-steps acid dissociation constants of L-ascorbic acid (AA) were determined through the changes in the effective electrophoretic mobility in capillary zone electrophoresis. Although ascorbic acid is oxidatively degradable in an aqueous solution, especially in alkaline conditions, the effective electrophoretic mobility of AA was successfully measured in the pH range between 1.87 and 11.97. In the analysis of the first-step acid dissociation constant (pKa1) at weakly acidic pH conditions, a coated capillary with 1,3-propanesultone and a pressure-assist were utilized to detect anionic AA. In the analysis of the second-step acid dissociation constant (pKa2) at weakly alkaline pH conditions, AA was successfully detected at the pH range up to 11.97 with the help of Cu catalyst to remove the dissolved oxygen in the separation buffer. Acid dissociation constants were independently determined as pKa1 = 4.15±0.01 and pKa2 = 12.07±0.04 by non-linear least-squares analyses. AA did not fully dissociate at the weakly alkaline pH range, and the effective electrophoretic mobility of the dianion form of AA was extrapolated for the analysis

Analysis of Acid Dissociation Equilibrium of Bupropion by Capillary Zone Electrophoresis After Degradation Under Heat

An acid dissociation constant (pKa) of bupropion has been determined in an aqueous solution by capillary zone electrophoresis (CZE). Electrophoretic mobility of bupropion was measured by CZE over its acid-dissociating pH range, and a pKa value has been determined as 8.75±0.02 (mean ± standard error, ionic strength: 0.010 mol dm−3). Although bupropion degrades under heated and alkaline conditions and the degraded species from bupropion coexist in the aqueous solution, the pKa value of bupropion can also be determined by CZE with the residual bupropion without any interference from the degraded species. The pKa value determined after the heat-degradation was 8.74±0.02; the result agreed well with the one determined with the freshly prepared solution. Utilization of the separation characteristics of CZE has been demonstrated with bupropion for the analysis of the fast equilibrium of acid-base reaction under the coexistence of the degraded species

Migration Behavior of Carbon Nanotube in Capillary Electrophoresis with Sodium Dodecyl Sulfate and Water-Soluble Nonionic Polymer

Single-walled carbon natobube (SWCNT) was dispersed in an aqueous surfactant solution, and the dispersion of CNT was analyzed by capillary electrophoresis (CE). The dispersion was evaluated through a broad peak of the electropherograms, while the aggregation of the CNT was attributed to the shot signals in the electropherograms. Water-soluble nonionic polymer was also added in the separation buffer to examine the dispersion in the surfactant solution as well as to control the migration behavior. Polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone were examined. The electrophoretic mobility of the broad peak got smaller with increasing concentrations of the polymer examined. The theoretical number of plates of the broad peak was improved by the addition of the polymer. Moderately broad peak was preferable for the dispersion of the SWCNT in the surfactant solution

Dispersion of Graphene in an Aqueous Solution with Poly(sodium 4-styrenesulfonate) Monitored by Capillary Electrophoresis

Graphene was dispersed in an aqueous solution with poly(sodium 4-styrenesulfonate) as a dispersant. The charge of the graphene came to be apparently negative by the adsorption of poly(4-styrenesulfonate) ion (PSS). Two kinds of PSS were examined: the average molecular masses of 70,000 and 1,000,000 (PSS 70,000 and PSS 1,000,000, respectively). Capillary electrophoresis was used to evaluate the dispersion of the apparently anionic graphene in an aqueous solution. A broad signal corresponding to the dispersed graphene was detected in the electropherograms. The effective electrophoretic mobility of the dispersed graphene was somewhat larger at higher concentrations of PSS 70,000, suggesting that the adsorbed amount of PSS 70,000 increased. Even when the separation buffer did not contain PSS, the broad signal of the anionic graphene was still detected. The peak height and/or the peak area, as well as the effective electrophoretic mobility of the graphene decreased little at the reduced applied voltages, i.e., at longer separation/detection time. Therefore, the adsorption of PSS is irreversible or the desorption of PSS from the graphene surface is very slow. Accordingly, the dispersed graphene with PSS would be separated from the matrix PSS by the electrophoretic separation

Kinetic Analyses of Two-Steps Enzymatic Oxidation from Hypoxanthine to Uric Acid with Xanthine Oxidase by Capillary Electrophoresis/Dynamic Frontal Analysis

Two steps of enzymatic oxidations from hypoxanthine to uric acid with xanthine oxidase (XOD) were kinetically analyzed by capillary electrophoresis/dynamic frontal analysis. When a substrate solution of hypoxanthine was introduced into a capillary with a separation buffer containing XOD, the enzymatic reaction continuously proceeded during the electrophoresis and a product of xanthine was continuously resolved from the substrate zone. A plateau signal of the product xanthine was detected based on the constant reaction rate with XOD. The plateau height was directly related with the reaction rate, and a Michaelis-Menten constant KM,HXA was successfully determined as 770±40 μmol L−1. When xanthine was used as a substrate, a slope response of uric acid was obtained because of the low concentrations of the substrate and its significant decrease. However, the Michaelis-Menten constant was successfully determined by using the initial reaction rate, and a Michaelis-Menten constant of KM,XA was determined as 85±6 μmol L−1

Capillary Electrophoretic Characterization of Carbon Nanodots Prepared from Glutamic Acid in an Electric Furnace

Carbon nanodots (CNDs) prepared from glutamic acid or glutathione in an electric furnace were characterized by capillary electrophoresis. Two major peaks were detected in the electropherograms by capillary zone electrophoresis, corresponding to anionic and less-charged CNDs. The effective electrophoretic mobility of the anionic CND formed from glutamic acid was almost identical over neutral to weakly alkaline pH range, and the CND would not contain significant amount of amino group. On the other hand, the effective electrophoretic mobility tended to decrease with decreasing pH at weakly acidic pH conditions, suggesting the functional groups of carboxylate moiety on the anionic CNDs. Dodecyl sulfate ion was added in the separation buffer to give anionic charge to the less-charged CND by adsorption. However, the anionic charge induced was little, and the dodecyl sulfate ion was not likely adsorbed on the less-charged CND and the CND would be hydrophilic

A Practice of the Laboratory Course Using a Personal Spectrometer and Learning Management System

学生実験は，限られた授業時間，実験作業スペース，限られた数の機器など，いくつかの制約の下で行われている。これらの制約を緩和するために，予習用のコンテンツを学習管理システム（LMS）でオンライン公開した。2017年度前期より，学生一人ひとりが利用できる計測機器とタブレットを導入した。その後，アプリの改良を重ね，貸出用のタブレットではなく，学生が自分のスマートフォンを利用できるようになった。2022年度前期には，学生実験に一通りの方法が確立した。また，今後の改良点を模索するためにも学生実験終了後にアンケートを取った。本稿では，学生実験の概要，アンケート結果について報告する。Laboratory courses are conducted under some constraints, such as limited class hours, insufficient experimental workspace, and limited numbers of instruments. We released online preparatory content on the learning management system (LMS) to alleviate these restrictions. We introduced a measurement device and a tablet available to each student in the first semester of 2017. The measurement application has improved since then, and now students can use their own smartphones instead of tablets. An appropriate method for our laboratory course was completed in the first semester of 2022. In addition, a questionnaire was taken for further improvements after the laboratory course was finished. In this paper, we report the outline of the laboratory course and the questionnaire results

Track-etched membrane-based dual-electrode coulometric detector for microbore/capillary high-performance liquid chromatography

The electrochemical flow cell containing track-etched microporous membrane electrodes was applied to a dual-electrode coulometric detector for microbore/capillary HPLC with a small injection volume and low eluent flow rate. The proposed flow cell with a 0.1-mm diameter inlet channel gave a detection volume of 0.08 nL per electrode, which was determined by the eluent flow through the electrode. For the dual-electrode detector, the calculated volume was 0.24 nL. The efficiency of electrooxidation of L-ascorbic acid increased as the flow rate decreased and was close to 100% when the flow rate was below 50 µL min−1, which is a common flow rate in microbore or capillary liquid chromatography. Catecholamines, such as noradrenaline, adrenaline, and dopamine, were detected by total conversion with two-electron oxidation in the potential range from 0.8 to 1.0 V vs. Ag/AgCl after separation with a microbore column. These peaks were accompanied by corresponding cathodic peaks derived from quasi-stable electrooxidation products of the catecholamines. The detection limits of noradrenaline, adrenaline, and dopamine were 0.1, 0.1, and 0.2 μM, respectively. The RSD values for five replicate measurements of 5.0 μM of these compounds were 0.9%, 0.7%, and 1.5%, respectively. Coulometric detection was also demonstrated by determination of catecholamines in pharmaceuticals

Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis

Acid dissociation constants (pKa) of 9 kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light-exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pKa values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pKa values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pKa values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pKa values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pKa values of such analogues as possessing carboxylic acid moiety