Riichiro Nakajima

Microchip capillary electrophoresis using on-line chemiluminescence detection.

Chemiluminescence detection was used in capillary electrophoresis integrated on a microchip. Quartz microchips have two main channels and four reservoirs. Dansyl-lysine and -glycine were separated and detected with bis[(2-(3,6,9-trioxadecanyloxycarbony)-4-nitrophenyl]oxalate as peroxyoxalate chemiluminescent reagent. These dansyl amino acids came into contact with the chemiluminescence reagent to produce visible light at the interface between the separation channel and chemiluminescence reagent-containing reservoir. The detection limit (S/N = 3) for dansyl-lysine was 1 x 10(-5) M, which corresponded to the very small mass detection limit of ca. 0.4 fmol. However, the concentration sensitivity in the present system was approximately two orders of magnitude lower than that in the conventional capillary electrophoresis-chemiluminescence detection system. The relative standard deviations of migration time and peak height for dansyl-lysine were 4.2 and 4.5%, respectively. A channel conditioning before every run and an appropriate control of voltages were needed for the reproducible results. The present system had advantages in rapid separation time (within 40 s), small (several 10 pI) and accurate sample injection method using a cross-shaped injector, and simplification and miniaturization of the detection device.

Separation and determination of phenolic compounds by capillary electrophoresis with chemiluminescence detection.

Phenolic compounds were analyzed by means of capillary electrophoresis with chemiluminescence detection. Peroxyoxalate chemiluminescence reagent was used together with dansyl chloride as a labeling reagent. The reagent concentrations, the labeling procedures, and the performance of chemiluminescence detection cells were examined for sensitive detection of phenolic compounds. Six kinds of phenolic compounds (phenol, 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, and 2,4,6-trichlorophenol) were determined over a range of three orders of magnitude with detection limits of the order of 10(-7) M; their detection limits were ca. 10 times as low as those obtained by an ordinary fluorescence detector. A running buffer solution containing sodium dodecylsulfate and acetonitrile provided satisfactory results in the separation of 15 kinds of phenolic compounds. The combination of capillary electrophoresis with chemiluminescence detection and column concentration procedure supported the possibility that the present system could be applied to real samples such as surface and reused waters.

Compact detection cell using optical fiber for sensitization and simplification of capillary electrophoresis-chemiluminescence detection

Abstract A new and simple chemiluminescence detection cell using optical fibers was designed for capillary electrophoresis. The cell was easily combined with capillary electrophoresis equipment and the system was operated without any tedious procedures. Luminol chemiluminescence was adapted for use with this detection cell. Hydrogen peroxide and catalysts were examined in detail with respect to their usage and concentration using the cell. The detection limit for luminol was 5.0·10−10 M (14 amol; S/N=3), which was the most sensitive result reported so far. Also, a mixture of glycine, glycylglycine and glycylglycylglycine, which was labeled with isoluminol isothiocyanate, was subjected to the present system. They were sensitively detected and were baseline separated.

On-line chemiluminescence detection of proteins separated by capillary zone electrophoresis

Abstract In a capillary zone electrophoretic (CZE) experiment in phosphate buffer (pH 3.5), Eosin Y was found to migrate together with protein in a capillary tube as a supramolecular complex. This finding gave the possibilities not only of overcoming the problem of protein determination but also of measuring the Eosin Y comigrating with protein by a chemiluminescence (CL) method with high sensitivity. Labelling of protein with dyestuff was achieved simply by mixing the protein and a dyestuff. On-line CL detection of the protein separated by CZE was feasible by measuring the CL intensity of a bis(2,4,6-trichlorophenyl) oxalate (TCPO)-H2O2-dyestuff system by means of an interface between CZE and CL detection. Several xanthene dyestuffs including Eosin Y were examined and Rose Bengal was found to be more sensitive than Eosin Y used in previous work. Using the present method, 5 · 10−7−1 · 10−4 mol dm−3 of bovine serum albumin (BSA) could be determined using Rose Bengal in about 20 min with a detection limit of 2 · 10−7 mol dm−3 (signal-to-noise ratio = 3), corresponding to 4 fmol of BSA. The results for fifteen kinds of proteins including BSA are reported.

Preparation of Highly Photosensitizing Liposomes with Fullerene-Doped Lipid Bilayer Using Dispersion-Controllable Molecular Exchange Reactions

Fullerene-containing liposomes with high photosensitization ability were prepared. Disaggregated fullerenes were efficiently injected into the bilayer of liposomes by a phototriggered molecular exchange reaction. These liposomes showed far higher photoreactivity than liposomes thermally produced by heating and microwave irradiation. This result indicates that control of self-aggregation of fullerene leads to a high quantum yield for the photoreaction because of the suppression of self-quenching of photoexcited fullerenes.

Chemiluminescence detection of heme proteins separated by capillary isoelectric focusing

Chemiluminescence detection was combined with capillary isoelectric focusing to perform protein analysis with high sensitivity. Luminol-H2O2 chemiluminescence was utilized, and heme proteins such as cytochrome c, myoglobin and peroxidase were analyzed. The proteins were focused by use of Pharmalyte 3-10 as ampholytes. Hydroxypropylmethyl-cellulose was added to the sample solution in order to easily reduce protein interactions with the capillary wall as well as the electroendoosmotic flow. The focused proteins were transported by salt mobilization to chemiluminescence detection cell equipped with an optical fiber. The present method showed significantly high sensitivity and wide dynamic range; the detection limit for cytochrome c was 6 x 10(-9) M and the linear dynamic range was greater than two-orders of magnitude (up to 2 x 10(-6) M).

Migration behavior of dyestuff-containing liposomes in capillary electrophoresis with chemiluminescence detection

Abstract Various types of dyestuff-containing liposomes were prepared from eosin Y or rhodaminine B, dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylcholine–dipalmitoylphosphatidylserine mixtures, and buffer solutions (trapped solutions) differing in chemical species, buffer concentrations, and pH. The dyestuff-containing liposomes were subjected to capillary electrophoresis with chemiluminescence detection, and the effects of the constituents on liposome properties were examined from the obtained electropherogram. Two peaks were typically recorded on the electropherograms; one was due to dyestuff entrapped in the liposomes and another was due to free dyestuff in the bulk solution. The changes of retention times and ratios of the two peaks easily and rapidly offered useful information as to permeability and surface charge of the liposome membranes.

Miniaturization of batch- and flow-type chemiluminescence detectors in capillary electrophoresis

Glass and PTFE tubes as detection cells were put in small light-tight boxes to achieve miniaturization of batch-and flow-type chemiluminescence detectors for capillary electrophoresis. These light-tight boxes which included a detection cell and a photosensor module were successfully designed. In the batch-type detector using a glass tube as a detection cell, the influences of a repeated injection of sample and a reagent volume of the detection cell on chemiluminescence intensity were examined in detail. By using 3.8 mm I.D. glass tube including 400 microl chemiluminescence reagent solution, the chemiluminescence peaks were reproducibly observed for the repeated injection experiment up to the eight injection with each run time of 3.0 min. Dansyl-Trp was determined over the range 3 x 10(-8)-1 x 10(-5) M with the detection limit of 0.43 fmol (S/N=3). In the flow-type detector using a PTFE tube as a detection cell, both ends of the PTFE tube were connected to three-way joints; a chemiluminescence reagent solution was delivered into the cell and a capillary was inserted through one of the joints while an electrode was inserted through the other one. Dansyl-Trp was determined over the range 1 x 10(-7)-1 x 10(-5) M with the detection limit of 1.3 fmol (S/N=3). By using the compact flow-type detector, a mixture of dansyl-amino acids was separated and detected in micellar electrokinetic chromatography mode.

Consideration on peak shape in a batch-type chemiluminescence detection cell for capillary electrophoresis

Peak areas, peak heights, and apparent theoretical plate numbers were examined as a function of sample injection times by use of the batch-type CL detection cell. Comparing the experimental data with those obtained by absorption detector, some considerations were carried out about the peak shape. The peak shape in CL detection was influenced by not only concentration distribution of sample in a sample zone but also sample diffusion and CL reaction at the capillary outlet. The sample injection time of ca. 35 s was recommended for the present CE with CL detector. The injection time much influenced peak shape as well as sensitivity in the CL detection cell.

Separation Behavior of Biological Constituents Having cis-Diol Groups through Interactions with Phenylboronic Acid Sites Introduced on the Inner Wall of a Fused-Silica Capillary

Phenylboronic acid sites were introduced onto the inner wall of a fused-silica capillary through the polymerization of 3-acrylamidophenylboronic acid after activating the wall by a silane coupling reagent. The phenylboronic acid-modified capillary was examined from various viewpoints of separation chemistry. Nucleosides having cis-diol groups were mainly used as model samples. The phenylboronic acid-modified capillary successfully separated a mixture of nucleosides, for example, an adenosine and deoxyadenosine mixture, in capillary electrophoresis. The separation was based on an interaction between the cis-diol groups and the phenylboronic acid sites on the capillary wall. The cooperation of borate migration buffer with the modified capillary could improve the resolution. The separation of a D- and L-glucoses mixture was also indicated with the coexistence of β-cyclodextrin in electrophoresis using a modified capillary. Furthermore, mixtures of nucleosides were separated with satisfactory results by chrom...