[in Japanese]

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This review outlines the fundamental characteristics of micellar electrokinetic chromatography (MEKC), and then describes recent advances in MEKC achieved in the authors' laboratory toward highly sensitive detection and a versatile analytical capability. The former concerns (1) the characteristics of MEKC, such as the separation principle, chromatographic parameters, selectivity and resolution, thermodynamic parameters, retention index, and band broadening; (2) the factors affecting the selectivity, such as the surfactant structures, temperature, pH and various additives. The latter describes (3) the on-line sample concentration for improving the concentration sensitivity in MEKC and (4) the on-line coupling of MEKC with mass spectrometry (MEKC-MS). Several techniques for neutral sample stacking are presented and discussed from the viewpoint of concentration mechanisms. A new concept for sample concentration, named “sweeping”, is introduced with the proposed mechanism. Sample stacking can give more than 100-fold increases in the concentration sensitivity and sweeping a few thousand-fold. MEKC-MS can be performed by using high-molecular-mass surfactants and an electrospray ionization interface, an atmospheric pressure chemical ionization interface with low-molecular-mass surfactants, and a partial-filling technique to avoid the entrance of surfactant molecules into the interface.

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Development of analytical methods sensitive to interfaces is an essential requirement to elucidate specific behaviors of molecules at liquid-solid and liquid-liquid interfaces. A resonant second harmonic generation spectroscopy is newly developed and applied to analysis of molecules at interfaces. Analysis by electrochemical STM and time-resolved total internal reflection fluorescence spectroscopy is also described. Complexation reactoins and molecular recognitions at liquid-liquid interfaces are analyzed by measurements of surface tension and ion transfer polarogram, and unusual selectiveity is achieved using suitable host molecules.

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Lanthanide complexes have been successfully developed as fluorescence labels for the highly sensitive detection of various biological molecules with time-resolved fluorometric measurements. The applications of lanthanide fluorescence labels for bioassays, such as time-resolved fluoroimmunoassay and DNA hybridization assay, have been extensively investigated and have attracted the interest of many researchers. A time-resolved fluorescence measurement permits easy distinction of a specific fluorescence signal of long-lived labels from short-lived background fluorescence present in most biological samples, and also obviates problems associated with scattering light of the optical components. In the past several years, many new fluorescent lanthanide complexes were synthesized and developed as fluorescence labels for bioassay, and new practical systems of time-resolved fluoroimmunoassay and DNA hybridization assay were developed using these new labels. This paper reviews the developments and applications of lanthanide fluorescence labels as well as time-resolved fluoroimmunoassay and DNA hybridization assay in recent years.

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We have developed a highly sensitive fluorescent-derivatization reagent, 2-(2, 3-anthracenedicarboximido) ethyl trifluoromethanesulfonate (AE-OTf), for carboxylic acids. The reagent has made it possible to detect fatty acids at sub-fmol levels. ¹H-NMR studies of some AE-O-esters have suggested the possiblity of chiral discrimination of chiral carboxylic acids having chiral centers at a remote position from their carboxyl groups by labeling with chiral AE-OTf analogues. We prepared some chiral AE-OTf analogues and studied the discrimination of their diastereomeric derivatives of some branched fatty acids having a chiral methyl branch at the C2 C12 position. After derivatizing with these reagents, it was possible to separate them on an ODS column below 0°C and to discrimine most of them by ¹H-NMR. By fluorometric detection it was possible to detect them at fmol levels. These methods were successfully applied to determine the absolute configuration at the C15 position of a novel branched-chain fatty acid of a ceramido isolated from the Epiphytic Dinoflagellate, Coolia monotis, by using 80 μg of the ceramido.

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Threading intercalator is a DNA ligand, whose major substituent group connected to the intercalating part is required to go through the adjacent base pairs of double-stranded DNA when the ligand becomes bound. The situation is typically fulfilled with the N, N'-disubstituted imide derivatives of naphthalene-1, 4, 5, 8-tetracarboxylic acid (N, N-disubstituted naphthalene diimide derivatives). In a complex with double-stranded DNA, the two substituents in a threading intercalator are projecting out in the major and minor grooves of DNA. This configuration results in kinetic stabilization of the formed complex, because the threaded bulky substituents can act as an anchor to prevent the ligand molecule from a facile dissociation from the DNA strand. We have been developing new analytical reagents for DNA by taking advantage of these peculiar characters of threading intercalation. As a typical threading design, three intrinsically intercalating molecular units were linearly connected by aminoalkyl chains to give trisintercalators. The resultant ligand seemed to prefer binding to the “labile” portion or sequence in the double stranded DNA where the strand is more susceptible to breathing or base-pair disruption. In another molecular design, thymine bases were introduced in the two N, N'-arms of naphthalene diimide. This ligand bound preferentially to a non-alternating continuous adenine sequence of single-stranded RNA due to the formation of hydrogen bonding between the thymine in the ligand and the adenine in the single strand RNA. Due to a similar A-T base pair forming interaction, this ligand stabilized the bulged duplex structure of oligonucleotide d(GCGAAACGC), for which the hairpin structure is more stable under ordinary solution conditions. The naphthalene diimide ligand carrying ferrocene parts on its N, N'-substituent arms allowed a highly sensitive electrochemical detection of double-stranded DNA. This provides a promising method for selectively detecting and quantitating target genes of interest. The principle is based on the redox current obtained from the ligand concentrated on the electrode, where the electrode-supported probe strand and the complementary target strand in solution hybridize to form a double strand. On the other hand, such a double stranded DNA-ligand complex on the electrode can be deemed as a pseudo-polyferroocene strand, since the ferrocene moieties are arranged along a single molecular chain of double-stranded DNA. This configuration of ferrocene units on the electrode gives a unique electrochemical behavior in enhancing the electron transfer between the reduced glucose oxidase and the electrode. Cyclic bis-threading intercalators are expected to form a doubly intercalated complex with double-stranded DNA offering a catenated structure. A cyclic naphtalene diimide derivative carrying a ferrocene unit within the macrocycle should find an application in the electrochemical detection of a base-mismatched DNA strand.

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A new fluorescent reagent, N, N'-(4, 7, 10-trioxatridecane-1, 13-diyl)bis(anthracene-9-carbonamide) (5), and its analogues(14 and 6), which have two anthracene moieties at both terminals of linear polyethers, were synthesized. Their complexation behavior with alkaline earth cations was investigated by fluorescence spectrometry, fluorescence lifetime, and ¹H NMR spectrometry. When metal ions were added to these reagent solutions, fluorescence spectral changes were observed. In particular, 5 showed a drastic change in the fluorescence spectra accompanied by metal ion binding. The fluorescence spectra of 5 completely changed from that of monomer to excimer emission. All reagents formed a 1 : 1 complex with alkaline earth cations. The ¹H NMR spectra of 5 with alkaline earth cations suggested the stacking of two anthracene moieties with each other. However, the interactions between two anthracenes were weak on the ground state for the other compounds (14 and 6).

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A new homogeneous liquid-liquid extraction method based on pH-dependent phase separation phenomena was developed using a water/pyridine/ethyl chloroacetate ternary solvent system. The optimum conditions in the procedure were determined as follows: in order to obtain the water-immiscible small phase (approximately 120 μl) of ethyl chloroacetate, hydrochloric acid ([HCl]_T=1.44 mol dm^-3) was added to a homogeneous aqueous solution which consisted of water, ethyl chlorolacetate (1 cm³: 2.0 vol%) and pyridine (5 cm³: 12.0 vol%), where the final total volume was 50 cm³. A concentration factor of 10000 (i. e. 50 cm³→ 5μl) was achieved by adjusting the initial volume of ethyl chloroacetate. The compound α, β, γ, δ-tetrakis(4-carboxyphenyl)porphine (TCPP) was selected as an extracting chelate reagent. In the extraction of TCPP and metal-TCPP complexes, when the ratio ( V_w/ V_o) of the volume in the water phase ( V_w) to that in the sedimented phase ( V_o) was 500, the distribution ratio (D) and extraction percentage (E%) were TCPP (14700, 96.7%), Cu-TCPP (7440, 93.7%), Zn-TCPP (22200, 97.8%), Mn-TCPP (1070, 68.1%) and Co-TCPP (8760, 94.6%), respectively. This extraction method was applied as preconcentration in HPLC. The simultaneous complex formation condition between TCPP and each metal ion { i.e., accelerator; [pyridine]_T = 0.25 mol dm^-3 and [Hg²⁺]_T = 2×10^-6 mol dm^-3, pH; 9.3, reaction time; 30 min at room temperature} and The HPLC condition of the metal complexes { i.e., column; ODS (4.6 mm i.d.×250 mm), mobile phase; the mixture (50 w/w%) of acetonitrile and acetic acid-sodium acetate aqueous solution (pH 3.5), flow rate; 0.5 cm³/min, detection wavelength; 420 nm } were selected, respectively. All calibration graphs were linear and passed through the origin over the concentration range of 5×10^-9 mol dm^-3 to 10^-7 mol dm^-3. The detection limit (3σ) reached 1×10^-10 mol dm^-3 for Cu(II) and Mn(II) and 5×10^-11 mol dm^-3 for Zn(II) and Co(II), respectively. Satisfactory results for the determination of four kinds of metal in river water (standard material in the Japan Society for Analytical Chemistry: JAC 0032) was obtained.

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An electrochemical detector for flow injection analysis (FIA) was designed and applied to the determination of residual chlorine. The detector consists of a working electrode (changeable solid electrode), a counter electrode (platinum wire) and a reference electrode {saturated calomel electrode (SCE)}. Evaluation of the detector was carried out using hexacyanoferrate (II). A linear relationship was obtained in the concentration range of 2×10^-7 to 2×10^-4 M. The relative standard deviation (RSD) for ten injections was less than 1%. The electrochemical behavior of hypochlorous acid was examined using cyclic voltammetry. At a gold electrode, a reduction wave of hypochlorous acid was observed at + 0.3 V vs. SCE. A linear calibration curve was obtained in the concentration range of 0.05 to 2.5 mg l^-1 when the gold electrode was applied to the FIA. The RSD was 2.1% for 1 mg l^-1 hypochlorous acid. The proposed method was applied to the determination of residual chlorine in tap water. The results agreed well with those obtained by the ο-tolidine photometric method.

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A collection method for anionic species on protonated chitin has been applied to the solid-phase spectrophotometric determination of trace copper. Copper is easily collected as its colored bathocuproinedisulfonate chelate anion on 20 mg of chitin from 20 cm³ of aqueous solution at pH 5.0. The reflection absorbance of the chitin phase at 484 nm was measured directly. The calibration curve was linear in the concentration up to 1 μg of copper on 20 mg of chitin. The relative standard deviation was 2.89% for 0.5μg of copper with 9 measurements. The tolerance limit for VO₃^-is 200-times in the concentration that of copper, and for Fe³⁺ and Al³⁺ it is 500-and 1000-times in the concentration of copper, respectively, when 5 mg of F^-is added. Common metal ions, reducing agents and inorganic anions do not interfere in the concentration range of 1000-to 10000-times that of copper. The present method can be applied to the determination of copper in biological standard samples.

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A simple solid-phase colorimetry of trace cobalt(II), nickel(II) and iron(II, III) was developed based on the L^*a^*b^* chromaticity diagram system. Metal complexes with 2-nitroso-5-(N-propyl-N-sulfopropylamino) phenol (Nitroso-PSAP) were preconcentrated in fine particles of anion-exchanger and collected on a membrane filter for subsequent measurements. A calibration curve for each metal ion was constructed by plotting the measured net values of +a^*(red), -a^*(green), +b^*(yellow) or -b^*(blue). The detection limits of cobalt(II), nickel(II) and iron(II) and iron(III), calculated from linearized calibration curves, were 0.1, 0.2, 0.3 and 0.4 μg, respectively. The proposed method can be applied to the simultaneous determination of cobalt(II) and nickel(II). The calibration plane of iron(II) and iron(III) has also made it possible to determine both species simultaneously.

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A chemical compound (A18CC) with a carbodithioate unit and an aza-crown ring in one intramolecule was synthesized, and reversed phase high performance liquid chromatography of the transition-metal chelates of A18CC was examined. The retention time of these A18CC chelates decreased along with an increase in the methanol fraction in the eluent. The retention time of the chelates decreased along with an increase in the concentration of alkali metal salt added to the eluent. The degree of the effect was Na⁺ > K⁺ >> NH₄⁺. Li⁺ and the alkaline earth metal ions hardly had any influence. Since the chelates had charge by taking in the alkali metal ion in the aza-crown ring, this behavior was generated.

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The HPLC resolution of the enantiomeric sn-1, 2- and sn-2, 3-diacylglycerol (DG) moieties of fish-oil triacylglycerols (TGs) having different fatty acid compositions was carried out on a chiral stationary phase, (R)-1-(1-naphthyl)ethylamine. The DGs were generated from TGs of tuna orbital, pomfret flesh and pacific saury flesh by partial Grignard degradation ; they were chromatographed as 3, 5-dinitrophenylurethanes. By an isocratic elution with n-hexane-1, 2-dichloroethane-ethanol 40 : 30 : 1 (v/v/v) as the mobile phase, the sn-1, 2 (2, 3)-DGs from tuna orbital and pomfret flesh were resolved into two clearly distinguishable enantiomer groups, although some peak overlappings between the enantiomers were observed in the latter sample. On the other hand, no clear enatiomer resolution was obtained for the saury flesh DGs. The poorer resolution was mainly caused by the existence of large amounts of long-chain monounsaturated fatty acids (20 : 1 and 22 : 1) in the molecules.

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Three lipases were immobilized on ODS - Sugar, which was prepared by coating sucrose mono-stearate (sugar ester) on octadecyl silica gel (ODS-silica). The enantioselective esterification of (R)- or (S)-1-phenylethanol with aliphatic acid was studied in the presence of immobilized lipase (ODS - Sugar-Lipase) in organic solvents by varying the lipase origin, reaction media and substrate structures. The ODS - Sugar - Lipase (LPL-311) showed both high catalytic activity and enantioselectivity for the esterification of (R)-1- phenylethanol with long-chain aliphatic acid in dry iso-octane. On the other hand, the ODS- Sugar- Lipase (M) showed both a high catalytic activity and enantioselectivity for the esterification of (S)-ibuprofen with hexanol in dry iso-octane. The enzymatic activity of lipases in organic media was remarkably enhanced by immobilization with the sugar ester. The reaction rates of the immobilized lipases were increased by around 100200-fold compared to that of the native lipases. The activity of the immobilized lipases has been retained for at least one month in refrigerator. The ODS- Sugar- Lipase showed greater activity for ester synthesis than other systems, such as the PEG-grafted lipase and the powder dispersion system. We believe that the ODS - Sugar adsorbed enzyme system can be widely applicable to other enzymes whose substrates are lipophilic.

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