This paper reviews recent 5-years application researches using online sample preconcentration (OSP) methods in capillary electrophoresis (CE) coupled with mass spectrometry (MS). CE-MS has excellent analytical features such as high-resolution separation, rapid analysis time, low sample consumption, and small consumption of organic solvents. Recently, low flow-rate interface for electrospray ionization has been developed and zmol-level sensitivity has been achieved. However, application of CE-MS has still been prevented due to small capacity of sample injection volume. OSP methods have been developed to address this issue and up to 5,000-fold sensitivity improvement was obtained so far. Various application has been carried out such as proteomics, metabolomics, and glycomics, and CE-MS attracts much attention as the highest-performance analytical tool for small-volume bioanalysis. Taking account of further progress in CE-MS, smart and practical application of OSP methods will be more important in various analytical fields.

Hyperbranched amine-modified capillary columns containing multiple terminal amine functional groups were prepared by repeating simple reactions of amine and diglycidyl. The retention behavior of some common inorganic anion samples (i.e. IO₃^-, NO₃^-, I^-) on the prepared columns was investigated by ion chromatography, and was then compared to the retention behavior using stationary phases reacted with other amines. The eluents contained sodium chloride in neutral eluent conditions, and sodium chloride and hydrochloric acid in acidic conditions were used as the mobile phases. The retention times of inorganic anions and hydrophobic samples increased with increasing generation of hyperbranched stationary phases. In addition, the anion-exchange capacities were determined by breakthrough curves.

Currently, nicotine is mostly analyzed by chromatography with pretreatment such as solid phase extraction (SPE). One of effective pretreatment techniques would be affinity extraction; however, there is no practical biomolecular affinity media available for SPE of nicotine. Molecular imprinting has been studied as a methodology for producing nicotine-selective synthetic affinity media, which relies on the formation of complex species between a target molecule (as template) and functional monomer; therefore, the selection of functional monomer is greatly important for obtaining molecularly imprinted polymers (MIPs) with high affinity and selectivity. In this study, itaconic acid (IA), which bears two carboxyl groups, was used as functional monomer in the synthesis of nicotine selective-MIPs. Other acidic monomers, such as methacrylic acid (MA), 2-(trifluoromethyl)acrylic acid (FM) and methyl itaconate (MI) were compared with IA to evaluate their usefulness as functional monomer. In chromatographic tests, the retention factor for nicotine on a MIP synthesized with itaconic acid (IP-IA16) was 40.4, which was 3.1 times that on a non-imprinted polymer (BP-IA16), while the retention factor on MIPs with the other monomers was 21.6 or less and was 1.1 times that on corresponding non-imprint blank polymers (BPs). In the selectivity test using cotinine, 3-methylpyridine and N-methylpyrrolidine as reference compounds, IP-IA16 showed the largest retention factor for nicotine, which was more than 4.4 times that of the other compounds, suggesting that nicotine-recognition sites were formed in MIPs by the molecular imprinting using IA as functional monomer.

In HPLC analysis of glycoprotein glycans, oligosaccharides released from glycoproteins are often derivatized with fluorescent tags for enhancing sensitivity and selectivity. However, existing analysis methods require multiple steps for removing excess reagents and are time consuming and sometime spoil the reliability in quantitative determination. Here, we developed an online sample cleanup procedure for the analysis of 7-amino-4-methylcoumarin (AMC)-labeled oligosaccharides. An NH₂-bonded silica packed short column (1 mm I.D. x 15 mm) and a Waters HLB^TM-packed column (4 mm I.D. x 5 cm) were used for online cleanup HPLC analysis with Amide80 and ODS columns, respectively. Optimized conditions enabled the direct injection of the diluted solution of the reaction mixture onto the chromatographic system without any prior removal of the excess labeling reagents. The method was successfully applied to the analysis of N-linked glycans released from some glycoprotein samples.

The effect of column structure and chromatographic conditions (flow rate, inlet pressure, and temperature) on separation behavior in low-temperature HPLC using pure liquid CO₂ mobile phase was investigated. Three types of packed columns (fully porous, small nonporous, and core-shell particles) and a monolithic column (fully porous) were used. The effect of temperature (-5 to -30°C), inlet pressure (5.3 or 6.3 MPa), and flow rate (1 or 2 mm/s) on chromatographic behavior was evaluated. The retention factors of the analytes (naphthalene, anthracene, and pyrene) were not affected by either inlet pressure or flow rate. Although the column temperature somewhat affected the separation efficiency, the typical relationship between temperature and separation efficiency was not observed. On the other hand, both inlet pressure and flow velocity affected the separation efficiency. In this study, the highest separation efficiency was achieved in the core-shell column with a flow rate of 1 mm/s and inlet pressure of 6.3 MPa. The best separation efficiency under this condition was 12 m in theoretical plate height (85 000 N/m) for pyrene at -30°C. In addition, the supplied liquid CO₂ was found to solidify in the column at low temperatures and its behavior also depends on both the chromatographic conditions (particularly for flow rate) and the column structure. The typical relationship between solidification and chromatographic behavior was not observed clearly.

This paper reports an on-line sample preconcentration by a sweeping technique based on the surface complexation between TiO₂ (titania) nanoparticles and phosphate groups in the capillary electrophoresis (CE) analysis of phosphopeptides. It is well-known that titania particles can trap phosphopeptides under an acidic condition, while under an alkaline condition they are eluted from the titania surfaces. In the sweeping via solid phase extraction using the titania nanoparticles, a phosphopeptide solution is injected as a long plug into a capillary filled with an acidic background solution. Due to the complexation onto the titania surfaces, the analytes in a long sample zone are swept by the titania particles to a narrow zone. This preconcentration technique was applied to the analysis of monophosphopeptide from β-casein. When an 1% titania dispersion and 100 ppm phosphopeptide solution was successively introduced into the capillary at the injection time of 30 s and 60 s, respectively, a sharp peak of the phosphopeptide was obtained by UV detection without any optical interferences of the titania. Comparing with a conventional CE analysis, a 22-fold sensitivity increase was achieved by the preconcentration effect.