Electrophoretic migration of an analyte in capillary electrophoresis (CE) reflects reaction dynamics of the analyte in solution. In affinity CE, an analyte of interest interacts with a modifier added in the separation buffer in fast equilibrium, and effective electrophoretic mobility of the analyte is contributed from its equilibrium species. Precise measurement of effective electrophoretic mobility allows analyzing the equilibrium. Analysis of equilibria under CE separation possesses several advantages against traditional analyses in homogeneous solution; coexisting substances including impurities and kinetically generated substances are resolved by CE from the equilibrium species of interest. Characteristics of the CE analysis have been applied to analyses of acid-base equilibria of degradable substances and ion-association equilibria in an aqueous solution. Since CE is operated in an open-tubular capillary, it is also suitable for the characterization of carbon nanoclusters such as graphene and carbon nanotube, and measurement of effective electrophoretic mobility helps characterization of nanoclusters. A novel analysis technique of capillary electrophoresis/dynamic frontal analysis (CE/DFA) has also been proposed for the analysis of such reactions as involving equilibria and kinetic reactions. In CE/DFA, kinetically generated product is continuously resolved from the equilibrium species, and a plateau signal would be detected when the reaction rate is constant. Michaelis-Menten constants have successfully been determined through the plateau height by CE/DFA. In this review, analysis and characterization methods utilizing reaction dynamics in a separation capillary are summarized.
π interactions have recently received considerable attentions due to principal factor governing molecular recognitions and self-assemble abilities, as accumulated in the database on proximate arrangements in structures of biological systems and organic functional materials. Therefore, further deep understanding and control of π interactions will greatly facilitate the development of new functional materials. Despite the importance of π interactions, they are still challenging to study because π interactions are much weaker than most other molecular interactions, such as hydrophobic interaction, hydrogen bonding, and electrostatic bonding. On the other hand, liquid chromatography (LC) is a powerful separation technique, which is able to distinguish the partition coefficients of solutes between the mobile and stationary phases, and can sensitively reflect the strength of molecular interactions. We investigated the properties of π interactions by developing new silica-monolithic capillary columns modified with carbon materials providing strong π interactions. In this focusing review, we introduce a few specific π interactions by columns modified with fullerenes and polycyclic aromatic hydrocarbons (PAHs), which showed strong π-π interactions due to spherical recognition and multiple CH-π interactions. Furthermore, π interactions can contribute to the separation of various samples, which are difficult to achieve by the available retention mechanisms. Briefly, we applied various π interactions to specific separation analyses, and we succeeded in separating halogenated compounds, H/D isotopologue pairs, and saccharides by effective π interactions. These results indicate that π interactions contribute to practical separation science, such as removal of environmental pollutants and quantitative determination of medicinal compounds.
We elucidated factors for the chiral conversion of α-lipoic acid enantiomers in α-lipoic acid-containing dietary supplements. Samples were cleaned up by the solid-phase dispersive extraction method using Oasis MAX and MCX as the solid-phase gel. The α-lipoic acid enantiomers were reciprocally converted by heating at 180°C, and finally became a racemate. The chiral conversion rate changed depending on sample purity, particularly the presence or absence of coexisting components in the dietary supplements. As candidates for coexisting components, neutral nonionic and highly polar substances were suggested, such as sugars. We found that chiral conversion was promoted by heating in the presence of glucose. Oftentimes, a relatively large amount of S-(–)-α-lipoic acid is detected in dietary supplements claiming to contain R-(+)-α-lipoic acid on the bottle label. We speculate that the proportion of S-form may have been increased by coexisting components such as glucose when heat treatment is performed during the manufacturing process.
A two-dimensional high-performance liquid chromatographic (2D-HPLC) system using the pre-column derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) has been developed for the determination of alanine (Ala), aspartic acid (Asp) and serine (Ser) enantiomers. For the precise determination of trace amounts of D-amino acids in complex matrices, a highly sensitive and selective method is required. In the present study, amino acids were derivatized with DBD-F for the sensitive fluorescence detection and were analyzed by a selective 2D-HPLC system combining a reversed-phase column (Singularity RP18, 1.0 x 500 mm) and an enantioselective column (Singularity CSP-001S, 1.5 x 250 mm). The established system was successfully applied to the chiral amino acid analyses of Japanese traditional amber rice vinegar, human plasma and human urine samples. In the Japanese traditional amber rice vinegar and human urine, relatively high amounts of all the target D-amino acids were observed (%D=5.8-37.7), while trace amounts of D-Ser and D-Ala were found in human plasma (%D=0.5-2.1).
To achieve highly sensitive analyses of phosphopeptides with simple experimental procedures in capillary electrophoresis (CE) and microchip electrophoresis (MCE), large-volume sample stacking with an electroosmotic flow pump (LVSEP) was performed in dynamically poly(vinyl pyrrolidone) (PVP)-coated capillaries to suppress the electroosmotic flow. In the analysis of monophosphopeptide (MPP) from bovine β-casein, good enrichments were attained with the sensitive enhancement factor (SEF) of 260 and 400 in CE and MCE, respectively, by using 0.2% PVP in 20 mM HEPES buffer (pH 9.0) in LVSEP.
2-Hydroxyglutarate (2-HG) was enantiomerically separated using copper(II) acetate and N,N-dimethyl-L-phenylalanine as chiral additives. These compounds formed diastereomeric complexes with 2-HG, which were successfully separated on an achiral (ODS) column. Several parameters, such as additive concentration, the type of organic modifier, and column temperature, were optimized. Using the optimal mobile phase (1 mM copper(II) acetate and 2 mM N,N-dimethyl-L-phenylalanine in a 10% aqueous methanol solution), 2-HG enantiomers were successfully separated in 15 min with a resolution of 1.93.
A novel spherical polymeric material has been introduced as a stationary phase in liquid chromatography (LC). Poly(benzoguanamine-co-melamine-co-formaldehyde) (BMF) was packed into a stainless-steel tube having 4.6 mm i.d., 150 mm length, as a particulate separation medium in LC. The retention behavior for polycyclic aromatic compounds (PACs) on the BMF phase was evaluated in reversed-phase LC and the trend was compared with that on a commercially-available octadecylsilica (ODS) phase. The BMF phase showed a unique molecular shape selectivity for PACs. It was clearly different from that on typical ODS phases. The BMF stationary phase more strongly retained "rod-like" molecules such as naphthacene than "square-like" molecules with a relatively large molecular weight. From the results, it was considered that the phenyl groups as stationary phase ligands were exposed on the surface of BMF particles, and these phenyl functionalities were apart from each other. Therefore, the BMF phase strongly retained "rod-like" analyte molecules that can simultaneously interact with more than one phenyl groups of the stationary phase.