In capillary and microchip electrophoresis, various on-line sample preconcentration techniques have been developed to improve the sensitivity. Among them, a large volume sample stacking with an electroosmotic flow EOF pump (LVSEP) features unique characteristics. LVSEP requires only a hydrodynamic sample injection into the whole capillary/microchannel and a constant voltage application, i.e., without polarity switching, and provides efficient sensitivity enhancement by the field-amplified sample stacking with a simple experimental procedure without loss of the effective separation length. In this review, a combination of LVSEP with a dynamic coating technique for analyzing phosphopeptides and metal-complexes, the development of a vacuum-drying method to immobilize polymer reagents onto the microchannel surface for the LVSEP analysis of cationic analytes, and a coupling of LVSEP with a field-amplified sample injection technique for highly-sensitive microscale electrophoretic analyses are presented.

As one of the microscale high performance liquid phase separation methods, capillary electrophoresis (CE) was introduced in around 1980. Although CE provides significant high resolution, only ionic species can be analyzed by CE. To overcome this limitation, micellar electrokinetic chromatography (MEKC) was developed, where neutral analytes as well as ionic ones can be separated. Since then, CE including MEKC have been used in various analytical fields. This paper describes the retrospective of MEKC development briefly from the view point of the author. Some examples of recently developed techniques, including online sample concentration methods in CE/MEKC and novel separation fields in HPLC, are also discussed.

Capillary electrophoresis (CE) is a high-resolution separation method. Coupled with sensitive detectors such as laser-induced fluorescence and mass spectrometers, CE can detect zmol-level biomolecules dissolved in a nL-order solution. However, μL-order sample solution must be prepared in a usual bioanalysis protocol, resulting in practical sample loss of 99.9 % or more in the CE analysis. Here, novel dual stacking methods were developed for increasing the injection capacity of CE to the level of μL-order. These methods typically preconcentrate the biomolecule by around 1000-fold, realizing trace analysis of glycans, metabolites, and drugs from 1–100 cells and tissue microsections. In this report, achievements on CE-based trace bioanalysis are introduced.

Antibody-drug conjugates (ADCs), which use antibodies as a means of recognizing and transporting target sites and carry specific small-molecule drugs, are attracting attention. However, since ADC cleaves the four disulfide bonds of Immunoglobulin G (IgG) and binds the drug via a linker, the number and distribution of the bound drug varies, and the drug efficacy also varies. In order to isolate only those with high drug efficacy, it is necessary to develop a precise analysis technique for ADC. In this study, we attempted to modify amino group-terminated silica gel particles (SiO₂-NH₂) with poly(ethylene glycol) (PEG) and aromatic compounds to develop a new separation medium for ADC analysis. In previous studies, PEG was used as a spacer to express hydrophilicity, excluded volume effect, and interaction with low-molecular-weight drugs entrapped in antibodies. The prepared media were packed in a column for liquid chromatography (LC), and the conditions under which IgG was completely eluted were investigated. From the results of LC evaluations, we confirmed the synthesis of SiO₂-PEG-BA. IgG was completely eluted under acidic conditions with 2-propanol added to the mobile phase. LC analysis of IgG modified with a benzene ring on a SiO₂-PEG-BA packed column showed increased retention compared to unmodified IgG, suggesting the possibility of selective separation of ADCs.

This paper describes the development of a rapid simultaneous HPLC evaluation method of related substances and optical purity of diltiazem (cis-(+)-(2S, 3S) form) and its formulations by employing a 3 μm ovomucoid conjugated chiral column. After optimization of the mobile phase, changing organic solvent species and its concentration, buffer species and concentration, buffer pH etc., successful simultaneous determination of its deacetyl form (diltiazem main impurity and degradation product) and a minor enantiomer (cis-(-)-(2R, 3R) form) was achieved within 2.5 min. The mobile phase used was a mixture of acetonitrile and 0.02 mol L⁻¹ ammonium acetate buffer (pH 6.0) (12.5 : 87.5). By employing the developed HPLC method with a 3 μm ovomucoid column, no minor enantiomer and 0.1 %-0.4 % deacetyl form were found in all diltiazem drug substances, its tablets and injections (freeze-dried formulations) (cis-(+)-(2S, 3S) form), and they were stored at a room temperature for more than 10 years, showing diltiazem and its formulations are very stable pharmaceuticals.

A two-dimensional electrophoresis method, involving involves isoelectric focusing in the first dimension and polyacrylamide gel electrophoresis in the second dimension, is commonly used for the separation and analysis of protein mixtures. However, conventional methods have some drawbacks such as complex experimental procedures, long analysis time, and high consumption of reagents. In this study, based on the knowledge gained from our previous research on digital electrophoresis, a cartridge device with heterogeneous functionalized gels arranged and integrated in a two-dimensional pattern was used. The fundamental evaluation using model proteins confirmed the feasibility of sample introduction using the developed devices, digital sieving electrophoresis, and digital isoelectric focusing using pH-buffered gels. Furthermore, the verification of two-dimensional digital electrophoresis using a cartridge device with multiple pH-buffered gels and molecular sieving gels demonstrated continuous two-dimensional digital separation by employing the single device.

Development of selective molecular recognition materials is an urgent issue for precise concentration regulations of each isomer with different environmental toxicity. Molecularly imprinted polymers are well-known artificial materials with selective molecular recognition abilities. Then, a direct approach to achieving selective recognition of halogenated aromatic compounds is synthesis of molecularly imprinted polymer by using highly specific intermolecular interactions with halogen groups. Halogen groups possess electron-deficient regions along the σ-bonding direction, and it enables the formation of weak and specific intermolecular interactions between the halogen group and the electron-rich π-conjugated surface (X-π interactions). However, the utilization of X-π interactions in the synthesis of molecularly imprinted polymers is still limited. In this study, we investigated the synthesis of MIPs based on X-π interactions and developed selective molecular recognition materials for di-halogenated benzenes.