Development of an ultra-micro sample injector for gas chromatography using an ink-jet microchip.

An ultra-micro sample injector for gas chromatography (GC) was developed. An ink-jet microchip, originally used for industrial recorder, was modified at the edge near to an orifice, and fixed into the GC. In order to evaluate the characteristics of this injector, a sample injector and a thermal conductive detector (TCD) were connected directly, while water was used as the test sample. The volume of the droplet, the interval time and the back-pressure to the ink-jet microchip were investigated. Within the range of 1 - 5 nL volume injected sample, the TCD response according to the amount of the sample volume (the volume of one droplet from the ink-jet microchip was about 1 nL) was obtained. A good reproducibility of the peak area was obtained to be about 1.0% of the RSD value. In order to compare the injection method of the ink-jet chip with that using a micro-syringe, the method using the ink-jet chip could introduce 1/1000 of the amount of the sample and gave reproducible results.

An integrated microfluidic device in polyester for electrophoretic analysis of amino acids.

A precolumn reaction chamber was integrated into a polyester microfluidic device with a miniaturized detection system. The reaction chamber was designed to be a zigzag channel, 70 microm in width, 8 mm in length, followed by a wider straight channel, 150 microm in width, 2 mm in length. The detection system is composed of an embedded light-emitting diode (LED), an integrated optical fiber, and a photomultiplier tube (PMT). A success in amino acid analysis using the integrated microchemical analysis device proved that the precolumn reaction chamber was compatible with the integrated detection system. Three kinds of amino acids, arginine, glycine, and phenylalanine, mixed and reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) in the precolumn reaction chamber to produce fluorescent products, were separated by micellar eletrokinetic chromatography (MEKC) and detected by LED-excited fluorescence. The detection limits for arginine, glycine, and phenylalanine were 1, 1, and 0.5 mM, respectively, which can be improved by further optimizations of the reaction system and detection system.

Spatial resolution improvement of scanning microscopy based on thermal lens spectroscopy with a total-internal-reflection arrangement.

In scanning microscopy by total internal reflection with thermal lens spectroscopy, its spatial resolution depends on the distance between the sample and a converging lens, which corresponds to the objective lens in an ordinary optical microscope. It was found that the resolution was best when the signal induced by the thermal lens effect was maximum. The distance was precisely adjusted by monitoring the signal intensity, and the resolution became twice better than that previously reported. Using a shorter focal-length lens, a resolution of 1.9 microm was attained.

A compactly integrated laser-induced fluorescence detector for microchip electrophoresis.

A simple and easy-to-use integrated laser-induced fluorescence detector for microchip electrophoresis was constructed and evaluated. The fluid channels and optical fiber channels in the glass microchip were fabricated using standard photolithographic techniques and wet chemical etching. A 473 nm diode-pumped laser was used as the excitation source, and the collimation and collection optics and mirrors were discarded by using a multimode optical fiber to couple the excitation light straight into the microchannel and placing the microchip directly on the top of the photomultiplier tube. A combination of filter systems was incorporated into a poly(dimethylsiloxane) layer, which was reversibly sealed to the bottom of the microchip to eliminate the scattering excitation light reaching to the photomultiplier tube. Fluorescein/calcein samples were taken as model analytes to evaluate the performance with respect to design factors. The detection limits were 0.05 microM for fluorescein and 0.18 microM for calcein, respectively. The suitability of this simple detector for fluorescence detection was demonstrated by baseline separation of fluorescein isothiocyanate (FITC)-labeled arginine, phenylalanine, and glycine and FITC within 30 s at separation length of 3.8 cm and electrical field strength of 600 V/cm.

Detection method for microchip separations.

The features of analytical systems utilizing microfluidic devices, especially detection methods, are described. Electrochemical detection (EC), laser-induced fluorescence (LIF), mass spectrometry (MS), and chemical luminescence (CL) methods are covered. EC enables detection without labeling and has been used in recent years because of its low cost and sensitivity. LIF is the most generally used detection method in microchip separations. Use of LED as an excitation source for fluorescence measurement was also developed for the purpose of miniaturization of the entire system, including detection and separation. Although MS enables highly sensitive analysis, the interface between MS and micro channels is still under examination. This review with fifty-two references introduces interesting detection methods for microchip separations. Related separation methods using microfluidic devices are also discussed.

Development of a scanning microscopy by total internal reflection coupled with thermal lens spectroscopy.

Non-destructive measurement of a small region on a solid/liquid interface is of great importance in physical chemistry and biochemistry, especially in the research of thin films and cell membranes. Optical methods for surface analysis with high lateral resolution are suitable methods for monitoring them. We now report a new scanning optical microscopic method to which total internal reflection coupled with a thermal lens technique was introduced. Its lateral resolution was estimated both experimentally and theoretically. To experimentally estimate the resolution, the grid patterns of thin photoresist films with well-defined lateral structures were measured. The experimental resolution was about 45 microm, which was almost same as the diameter of the excitation beam at a glass/sample interface. From this result, it was verified that this new scanning microscopy ideally worked.

Chiral separation with ligand-exchange micellar electrokinetic chromatography using a D-penicillamine-copper(II) ternary complex as chiral selector.

D-Penicillamine is demonstrated for the first time as a chiral ligand for the enantioseparation of dansyl amino acids based on ligand-exchange micellar electrokinetic chromatography (LE-MEKC). Copper(II) was used as the central ion in the ternary complex. The effect of surfactant on the resolution was significant. A concentration of 20 mM sodium dodecyl sulfate (SDS) was shown to be necessary for the separation. Other important parameters, such as the concentration ratio of D-penicillamine (D-PEN) to Cu2+, the kind of metal central ion, the type and pH value of buffer, were also investigated. N-Acetyl-D-penicillamine and L-valine (Val), with similar structure to D-penicillamine, were applied as their copper(II) complexes as chiral selector and the chiral recognition mechanism is briefly discussed. Under optimum experimental conditions, i.e., 20 mM NH4OAc, pH 6.5, a 2:1 concentration ratio of D-penicillamine to Cu(II), 4 mM CuSO4 and 8 mM D-penicillamine, the chiral separation of eight pairs of different dansyl amino acid enantiomers was accomplished with resolution ranging from 1.1 to 5.9. When L-PEN was used instead of D-PEN, reversal of the migration order was observed.

Cyclodextrin-modified monolithic columns for resolving dansyl amino acid enantiomers and positional isomers by capillary electrochromatography.

We describe beta- and gamma-cyclodextrins (beta- and gamma-CD)-modified monolithic columns prepared by sol-gel process and chemical modifications. The monolithic silica column was fabricated inside a fused-silica capillary with 100 microm inner diameter by sol-gel process. The monolithic silica matrix was chemically modified chiral selectors of beta- or gamma-CDs with a spacer of 3-glycidoxypropyltrimethoxysilane by on-column reactions. Gamma-CD-modified monolithic column has successfully been applied for the separation of dansyl amino acid enantiomers. Beta-CD-modified monolithic column has been used for the separation of the positional isomers of o-, m-, and p-cresols and the enantioseparation of racemates of benzoin and several dansyl amino acids by capillary electrochromatography, respectively. For the separation of neutral positional isomers, a positive electric field was applied. However, for the separation of negatively charged analytes, a negative electric field was applied at the inlet of column. The separation efficiency of 5.0 x 10(4) theoretical plates/m for dansyl-L-threonine was obtained at electric field strength of -300 V/cm in the mobile phase of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)-Tris/methanol (70/30) buffer at pH 7.0. L-enantiomers were eluted as the first peak. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pores.

Monitoring nano-flow rate of water by atomic emission detection using helium radio-frequency plasma.

Recently, high-performance nano-scale flow pumping systems have been developed for micro and miniaturized analysis systems. A novel device capable of measuring and monitoring nanoliter scale flow rates has been required for the further development of the pumping system. In this study, an atomic emission detector using helium radio-frequency plasma (RFP-AED) was used for the measurement of the nanoliter scale flow rate of water by quantitatively detecting the emission from hydrogen in the water molecules. Monitoring nano-flow rates of water in the range up to 1.0 microl min(-1), and the change in the flow rate by the indication of the ratio of the emissions of H (656.3 nm) and He (667.8 nm) were successful. At present, the lowest flow rate that could be determined reproducibly was 4 nl min(-1) calculated as five times the standard deviation of the background noise. Additionally, similar evaluations for the deviation of each flow rate by using the RFP-AED and a flow-injection system were produced.

Evaluation of lateral resolution of scanning surface microscopy by total internal reflection with thermal lens effect.

We have developed a novel method for in situ and non-destructive surface analyses, or a total internal reflection with thermal lens spectroscopy (TIR-TLS), which has sufficient sensitivity to monitor phenomena in thin films, such as lipid bilayers. In this study, we applied TIR-TLS to microscopy for surface analyses, and we experimentally obtained its lateral resolution using the edge of a chromium film made by a photolithography technique. The obtained resolution was 20 microm, which was 60% of the diameter of an excitation beam at the interface. The estimated resolution with a simple model agreed with the experimental one, and from this model, TIR-TLS microscopy has the same resolution as that of ordinary optical microscopy. The microscopy by TIR-TLS was applied to a sample whose contrast was too weak to be visually seen, and an image of the sample was obtained without any loss of resolution.

Comparison of enantioseparations using Cu(II) complexes with L-amino acid amides as chiral selectors or chiral stationary phases by capillary electrophoresis, capillary electrochromatography and micro liquid chromatography.

In this paper we report that Cu(II) complexes with L-amino acid amides were used as chiral selectors for enantioseparation by capillary electrophoresis, capillary electrochromatography (CEC) and micro liquid chromatography using chemically modified monolithic columns. The enantioselectivity, enantiomer migration order, and the performance have been compared when different chiral selectors were used in these modes. L-Enantiomers showed longer retention times than D-forms in both CEC and LC modes. However, it has interestingly been observed that the migration order of Dns-DL-Ser showed an exception in CEC using L-prolinamide-modified column that Dns-L-Ser was eluted as the first peak. On the basis of proposed structures of complexes in the chiral recognition, differences in migration orders and recognition mechanism were discussed.

Comparison of three different anionic surfactants for the separation of hydrophobic compounds by nonaqueous capillary electrophoresis.

The effect of the three different surfactants, sodium dodecyl sulfate (SDS), diethylhexyl sodium sulfosuccinate (AOT), and taurodexycholic acid sodium salt (STDC) on the nonaqueous capillary electrophoretic separations of hydrophobic compounds were compared with formamide containing 20 mM K2HPO4 as electrolyte solvent. Separations of all selected uncharged hydrophobic compounds, e.g., p-arylacetophones were shown to be strongly dependent on the kind of surfactant. The electrolyte containing 180 mM SDS provided the best result for the selected samples.

Chemically modified chiral monolithic silica column prepared by a sol-gel process for enantiomeric separation by micro high-performance liquid chromatography.

In this work a new type of chiral monolith silica column was developed for the chiral separation by micro high-performance liquid chromatography (micro-HPLC). The chiral monolith column with a continuous skeleton and a large through-pore structure was prepared inside a capillary of 100 microm I.D. by a sol-gel process, and chemically modified with chiral selectors, such as L-phenylalaninamide, L-alaninamide and L-prolinamide, on the surface of the monolithic silica column. Based on the principle of ligand exchange, these chiral monolithic columns were successfully used for the separation of dansyl amino acid enantiomers, as well as hydroxy acid enantiomers by micro-HPLC. The chromatographic conditions, the enantioselectivity and the performance of columns are discussed.

Photo-response assisted enantiomer separations on an azobenzene-modified gamma-cyclodextrin stationary phase in micro-HPLC.

The photo-responses of the retention and enantioseparation of several optical isomers were evaluated using an azobenzene-modified gamma-cyclodextrin stationary phase (Az gamma-CDSP) in micro-HPLC. UV light irradiation induced a decrease in the retention and the chiral selectivity for N-(3,5-dinitrobenzoyl)-1-phenylethylamine (DNBPEA) and N-(3,5-dinitrobenzoyl)-1-(1-naphtylethyl)amine (DNBNEA), while an increase was induced for dansylphenylalanine (DnsPhe) using a mixture of methanol and aqueous phosphate buffer as the mobile phase. No changes in the retention and the enantiomer separation of benzoin were observed with UV light irradiation. The retention behaviors were recovered by visible-light irradiation. It was speculated that the main factor of the change in the retention behavior was a change in the pi-pi interaction due to the azobenzene moiety of the stationary phase with photo-irradiation. Comparing the retention behavior before and after UV light irradiation, a suitable condition for obtaining a better resolution and enantiomer separation would be chosen using Az gamma-CDSP.