Fumihiko Kitagawa

On-Line Sample Preconcentration and Separation Technique Based on Transient Trapping in Microchip Micellar Electrokinetic Chromatography

This paper describes a novel on-line sample preconcentration and separation technique named transient trapping (tr-trapping), which improves the efficiencies of separation and concentration by using a partially injected short micellar plug in microchip electrophoresis. Although a longer separation length often provides a better resolution of complexed or closely migrating analytes, our proposed theoretical model indicated that a trap-and-release mechanism enables a short micellar zone, which was partially injected into the separation channel, to work as an effective concentration and separation field. Application of the tr-trapping technique to microchip micellar electrokinetic chromatography (MCMEKC) was performed on a newly fabricated 5-way-cross microchip by using sodium dodecyl sulfate and rhodamine dyes as test micelle and analytes, respectively. When the injection times of micelle (t(inj),M) and sample solution (t(inj),S) were 1.0 and 2.0 s, respectively, both the preconcentration and separation of the dyes were completely finished within only 3.0 s. At t(inj),S of 8.0 s, a 393-fold improvement of the detectability was achieved in comparison with conventional MCMEKC. The resolution obtained with tr-trapping-MCMEKC was also better than that with conventional MCMEKC in spite of the 160-fold shorter length of the injected micellar zone at t(inj),M of 1.0 s. These results clearly demonstrated that the tr-trapping technique in MCMEKC provides a rapid, high-resolution and detectability analysis even in the short separation channel on the microchips.

Highly sensitive chiral analysis in capillary electrophoresis with large-volume sample stacking with an electroosmotic flow pump

To improve the sensitivity in chiral analysis by capillary electrophoresis without loss of optical resolution, application of large-volume sample stacking with an electroosmotic flow pump (LVSEP) was investigated. Effects of the addition of cyclodextrin (CD) into a running solution on the LVSEP preconcentration was theoretically studied, where the preconcentration efficiency and effective separation length would be slightly increased if the effective electrophoretic velocity (v(ep,eff,BGS)) of the analytes was decreased by interacting with CD. In LVSEP-CD-modified capillary zone electrophoresis (CDCZE) and LVSEP-CD electrokinetic chromatography with reduced v(ep,eff,BGS), up to 1000-fold sensitivity increases were achieved with almost no loss of resolution. In LVSEP-CD-modified micellar electrokinetic chromatography of amino acids with increased v(ep,eff,BGS), a 1300-fold sensitivity increase was achieved without much loss of resolution, indicating the versatile applicability of LVSEP to many separation modes. An enantio-excess (EE) assay was also carried out in LVSEP-CDCZE, resulting in successful analyses of up to 99.6% EE. Finally, we analyzed ibuprofen in urine by desalting with a C₁₈ solid-phase extraction column. As a typical result, 250ppb ibuprofen was well concentrated and optically resolved with 84.0-86.6% recovery in LVSEP-CDCZE, indicating the applicability of LVSEP to real samples containing a large amount of unnecessary background salts.

Highly sensitive oligosaccharide analysis in capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump.

To obtain high sensitivity in capillary electrophoresis of oligosaccharide without reducing the high resolution with an easy experimental procedure, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was investigated. As a fundamental study, effect of the conductivity of a sample solution in LVSEP was examined. It was revealed that LVSEP was successfully carried out even in using a sample solution with the ionic strength of 150 μM and the conductivity ratio of 20, indicating a good applicability of LVSEP to the analysis of real samples containing salts. When glucose oligomer was analyzed as a model sample in LVSEP-capillary zone electrophoresis (CZE), all peaks were well resolved with decreasing only 5% of the peak-to-peak distance, which suggested 95% of the whole capillary could be used for the effective separation. In the analysis of maltoheptaose, a good calibration line with correlation coefficient of 0.9995 was obtained. The limit of detection was estimated as 2 pM, which was 500-fold lower than that in the conventional CZE. N-linked glycans released from three glycoproteins, bovine ribonuclease B, bovine fetuin, and human α(1)-acid glycoprotein were also analyzed by LVSEP-CZE. By the sample purification with a gel filtration column, further sample dilution to reduce the sample conductivity for LVSEP was not needed. All glycan samples were well concentrated and separated with up to a 770-fold sensitivity increase. The run-to-run repeatabilities of the migration time, peak height, and peak area were good with relative standard deviations of 0.1-1.3%, 1.2-1.7%, and 2.8-4.9%, respectively.

One-step immobilization of cationic polymer onto a poly(methyl methacrylate) microchip for high-performance electrophoretic analysis of proteins

Abstract One-step covalent immobilization of poly(ethyleneimine) (PEI) onto poly(methyl methacrylate) (PMMA) substrates was investigated to achieve an efficient separation of basic proteins in microchip electrophoresis (MCE). The PEI-treated PMMA chip showed the anodic electroosmotic flow and its rate was almost kept stable during 32 days with over 50 runs. This longer stability of the prepared microchip indicated that the loss of PEI was successfully suppressed by the immobilization through the covalent bond. Furthermore, the PEI modification onto the PMMA chip could apparently reduce the surface adsorption of cationic proteins. In the MCE analysis on the PEI-modified microchip, two proteins were successfully separated within 30 s only utilizing a separation length of 5 mm. While the migration time of the protein gradually increased during only four consecutive runs on an untreated PMMA chip, reproducible analyses were attained by using the PEI immobilized microchip. These results demonstrated that Coulombic repulsion force generated between cationic PEI and basic proteins could avoid the irreversible adsorption of the analytes onto the PMMA surface, which provided a high-performance analysis medium for biogenic compounds.

Micellar electrokinetic chromatography on microchips

This review highlights the methodological and instrumental developments in microchip micellar EKC (MCMEKC) from 1995. The combination of higher separation efficiencies in micellar EKC (MEKC) with high-speed separation in microchip electrophoresis (MCE) should provide high-throughput and high-performance analytical systems. The chip-based separation technique has received considerable attention due to its integration ability without any connector. This advantage allows the development of a multidimensional separation system. Several types of 2-D separation microchips are described in the review. Since complicated channel configurations can easily be fabricated on planar substrates, various sample manipulations can be carried out prior to MCMEKC separations. For example, mixing for on-chip reactions, on-line sample preconcentration, on-chip assay, etc., have been integrated on MEKC microchips. The application of on-line sample preconcentration to MCMEKC can provide not only sensitivity enhancement but also the elucidation of the preconcentration mechanism due to the visualization ability of MCE. The characteristics of these sample manipulations on MEKC microchips are presented in this review. The scope of applications in MCMEKC covers mainly biogenic compounds such as amino acids, peptides, proteins, biogenic amines, DNA, and oestrogens. This review provides a comprehensive table listing the applications in MCMEKC in relation to detection methods.

Label-free detection of amino acids using gold nanoparticles in electrokinetic chromatography-thermal lens microscopy.

To achieve label-free detection of amino acids in capillary-based electrokinetic chromatography-thermal lens microscopy (EKC-TLM), gold nanoparticles (GNPs), which possess the absorption around 500nm attributed to surface plasmon resonance (SPR), were added to the background solution (BGS). Since the SPR absorption of the GNPs exhibits a sensitive response toward environmental changes and the degree of aggregation, the sensitive detection of non-absorbing species is expected by using the GNPs in EKC-TLM (GNP-EKC-TLM). In the GNP-EKC-TLM analysis of glutamic acid (Glu), a sharp peak was observed when the GNPs were added to the BGS. The plot of the peak area of Glu against its concentration gave a good linear relationship and the limit of detection was estimated to be 25mug/mL. Furthermore, a baseline separation of lysine and Glu was successfully achieved. Thus, the EKC separation and label-free TLM detection of the amino acids can be realized only by adding the GNPs into the BGSs.

Polymer Microchip for Electrophoresis-Mass Spectrometry Fabricated by Hot Embossing and Low Temperature Direct Bonding

Integrated electrophoresis-mass spectrometry polymer devices were fabricated. The electrospray ionization (ESI) emitter tip structure was formed directly at the microchip electrophoresis (MCE) outlet. Since these devices enable negligible dead volume at the electrospray port, efficient spray of the sample necessary for high resolution mass spectrometry (MS) was realized. Stable spray was also achieved at the low flow rate (-0.1 muL/min) without additional pump. Low cost and high performance COP MCE-MS chip was fabricated by hot embossing and low temperature direct bonding.

Application of a partial filling technique to electrophoretic analysis on microchip with T-cross channel configuration

To achieve a high performance sample injection, we designed microchips with both T- and cross-type channel geometry (T-cross chip), which enables two sample solutions to be introduced into a separation channel simultaneously with desired volumes by a combination of gated and pinched injection techniques. The developed microchips were applied to microchip micellar electrokinetic chromatography (MCMEKC) using a partial filling (PF) technique. In the PF technique, to suppress the increase in the background noise due to the presence of a pseudostationary phase (PSP), e.g., ionic surfactant micelles in MCMEKC with mass spectrometric (MS) detection, in a background solution (BGS), the separation channel is partially filled with a PSP while the rest of the channel including the detection point is filled with the BGS without a PSP. As a result, both sodium dodecyl sulfate (SDS) micellar and sample solutions were simultaneously injected into the separation channel on the T-cross chip with a good repeatability of the injection lengths by applying programmed voltages, and a baseline separation of rhodamine derivatives was achieved within 40 s by MCMEKC, in which the micellar zone was partially injected with the PF technique. It was confirmed that the detection could be performed without any influence of SDS by the fluorescence imaging measurements. These results demonstrated that the developed T-cross chips will allow a combination of the MCMEKC separation with the MS detection by employing the PF technique for high performance analysis of biogenic compounds. In the PF?MCMEKC analysis, furthermore, the resolution of two rhodamines clearly increased from 1.30 to 1.73 with the increase of the injection length of PSP from 0.16 to 0.89 mm, whereas at the injection length of 1.47 mm the resolution decreased. Therefore, the selection of an optimal injection length on the T-cross chip is very important and the effects of the injection length on the separation processes in PF?MCMEKC were discussed on the basis of theoretical calculations.

Separation of fatty alcohol ethoxylates by capillary zone electrophoresis and micellar electrokinetic chromatography

Separation methods based on capillary zone electrophoresis and micellar electrokinetic chromatography were developed to characterize the distribution of ethylene oxide (EO) homologues in the fatty alcohol ethoxylates (FAEs). Prior to the separation, the FAEs were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMPTS) to allow CZE separation and UV detection. To prevent adsorption of cationic analytes onto the inner surface of the capillary and formation of micelles in CZE analysis, a lower pH background solution (BGS) containing a high concentration of acetonitrile was employed. Under optimal conditions, FMPTS-derivatized FAEs with an average EO number of 6 were completely separated within 11 min. For MEKC analysis of the FAEs, dodecyltrimethylammonium chloride (DTAC) was added to the BGS. In the presence of 30 mM DTAC in 10 mM phosphate buffer (pH 2.5) containing 20% (v/v) acetonitrile, superior oligomer separation of the FAEs containing up to 50 EO groups was achieved within 30 min with good analytical reproducibilities. Furthermore, the developed method was applied to the analysis of the FAEs in commercial products such as laundry detergent and fabric softener.

Separation of complex mixtures of fluorobenzoic acids by capillary electrophoresis

Fluorobenzoic acids are important intermediates in the synthesis of antibacterial drugs. Conditions for the separation of mixtures of twenty-five acids by CE have been optimized. A set of conditions with phosphate buffer (pH 3.1), successive multiple ionic-polymer layer (SMIL) coating capillary, and negative separation voltage provided a short time separation based on the difference in the acidic dissociation constant (pK(a)) of the sample acids. Addition of ACN to the separation solution improved the selectivity resulting in a broader distribution of migration times of the samples. The addition of tetradecyltrimethylammonium chloride below the CMC was also effective in changing the migration pattern of fluorobenzoic acids. Furthermore, practical utility was demonstrated through quantitative studies on the optimized condition including the durability of the SMIL coating.