Rhodamine B amine as a highly sensitive fluorescence derivatization reagent for saccharides in reversed-phase liquid chromatography

6-Rhodamine B amine functions as a highly sensitive fluorescence derivatization reagent for mono- and oligosaccharides; it reacts with the reducing end of saccharides under acidic conditions. The fluorescent derivatives of five monosaccharides can be separated within 25 min by reversed-phase liquid chromatography with isocratic elution. The detection limits (S/N = 3) for mono-, di-, and oligosaccharides are 7-51, 13, and 9-35 fmol/20 microl injection, which correspond to analyte concentrations of 35-255, 65, 45-175 nM, respectively. We have applied this derivatization method successfully to the analysis of the components of oligosaccharides in glycoproteins (ribonuclease B and fetuin) following their acidic or enzymatic hydrolysis. The results from these analyses are in good agreements with the reported values established previously.     

Power-free poly(dimethylsiloxane) microfluidic devices for gold nanoparticle-based DNA analysis

An extremely simple, power-free pumping method for poly(dimethylsiloxane)(PDMS) microfluidic devices is presented. By exploiting the high gas solubility of PDMS, the energy for the pumping is pre-stored in the degassed bulk PDMS, therefore no additional structures other than channels and reservoirs are required. In a Y-shaped microchannel with cross section of 100 microm width x 25 microm height, this method has provided flow rate of 0.5-2 nL s(-1), corresponding to linear velocity of 0.2-0.8 mm s(-1), with good reproducibility. As an application of the power-free pumping, gold nanoparticle-based DNA analysis, which does not rely on the cross-linking mechanism between nanoparticles, has been implemented in a microchannel with three inlets. Target 15mer DNA has been easily and unambiguously discriminated from its single-base substituted mutant. Instead of colorimetric detection in a conventional microtube, an alternative detection technique suitable for microdevices has been discovered-observation of deposition on the PDMS surfaces. The channel layout enabled two simultaneous DNA analyses at the two interfaces between the three laminar streams.     

Sample enrichment by using monolithic precolumns in microcolumn liquid chromatography

An on-line sample enrichment system was designed using monolithic precolumns in microcolumn LC. The monolithic ODS capillary columns were prepared via in situ sol-gel processes. The enrichment efficiency of the monolithic columns was tested by using phthalates as the analytes. The relative standard deviations (n = 6) for the retention time, peak area and peak height were between 0.4 and 1.2%, 0.9 and 5.5% and 0.4 and 3.9%, respectively. The system was linear (R2 > 0.99) within the working sample concentration and sample volume ranges. Comparing to 0.2 microl injection with a typical sample injector, the theoretical plate number of a same separation column was increased by 3-6-fold when the precolumn unit was used for sample injection. The recoveries of the analytes were between 88 and 120%, and the sample volume that could be injected into the system was increased up to 5000-fold. The limits of detection were improved by more than 2000-fold and were between 0.21 and 0.87 ng ml(-1) even with a UV absorbance detector. This system was applied to the determination of phthalates contained in laboratory distilled water and tap water samples.     

A solid phase extraction using a chelate resin immobilizing carboxymethylated pentaethylenehexamine for separation and preconcentration of trace elements in water samples

A chelate resin immobilizing carboxymethylated pentaethylenehexamine (CM-PEHA resin) was prepared, and the potential for the separation and preconcentration of trace elements in water samples was evaluated through the adsorption/elution test for 62 elements. The CM-PEHA resin could quantitatively recover various elements, including Ag, Cd, Co, Cu, Fe, Ni, Pb, Ti, U, and Zn, and rare earth elements over a wide pH range, and also Mn at pH above 5 and V and Mo at pH below 7. This resin could also effectively remove major elements, such as alkali and alkaline earth elements, under acidic and neutral conditions. Solid phase extraction using the CM-PEHA resin was applicable to the determination of 10 trace elements, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, V, and Zn, in certified reference materials (EnviroMAT EU-L-1 wastewater and ES-L-1 ground water) and treated wastewater and all elements except for Mn in surface seawater using inductively coupled plasma atomic emission spectrometry. The detection limits, defined as 3 times the standard deviation for the procedural blank using 500 mL of purified water (50-fold preconcentration, n = 8), ranged from 0.003 μg L⁻¹ (Mn) to 0.28 μg L⁻¹ (Zn) as the concentration in 500 mL of solution.     

Covalent conjugation of tetrameric bovine liver catalase to liposome membranes for stabilization of the enzyme tertiary and quaternary structures

Tetrameric bovine liver catalase (BLC) is unstable because of its dissociation into subunits at low enzyme concentrations and the conformational change of the subunits at high temperatures. In this work, for stabilization of BLC, the enzyme was covalently conjugated with liposome membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-glutaryl (NGPE). The NGPE, which was responsible for the BLC/membrane coupling, was altered from 0.05 to 0.2 in its liposomal mole fraction f_G. The catalase-conjugated liposome (CCL) with f_G of 0.15 showed the maximum number of the conjugated BLC molecules of 28 per liposome. The reactivity of CCLs to H₂O₂ was as high as that of free BLC at 25 °C in Tris–HCl buffer of pH 7.4. Among the CCLs, the catalyst with f_G of 0.15 was the most stable at 55 °C in its enzyme activity in the buffer because the appropriate number of BLC/liposome covalent bonding prevented the dissociation-induced enzyme deactivation. Furthermore, the CCL showed much higher stability at 55 °C than the free BLC/enzyme-free liposome mixture and free BLC at the low BLC concentration of 340 ng/mL. This was because BLC in the CCL was located in the vicinity of the host membrane regardless of the catalyst concentration, which could induce the effective stabilization effect of the membrane on the enzyme tertiary structure as indicated by the intrinsic tryptophan fluorescence analysis. The results obtained demonstrate the high structural stability of BLC in the CCL system, which was derived from the covalent bonding and interaction between BLC and liposomes.     

A fluorous tag-bound fluorescence derivatization reagent, F-trap pyrene, for reagent peak-free HPLC analysis of aliphatic amines

We have developed a novel pre-column fluorescence derivatization reagent for amines, F-trap pyrene. This reagent comprises a fluorescent pyrene moiety, an amine-reactive Marshall linker, and a fluorophilic perfluoroalkyl group known as fluorous tag. When the reagent reacts with aliphatic amines and amino acids to give fluorescent derivatives, the fluorous tag in the reagent is eliminated simultaneously. Therefore, excess unreacted reagents in the derivatization reaction solution still have the fluorous tag and could be removed by fluorous solid-phase extraction selectively before high-performance liquid chromatography (HPLC) analysis. By using this reagent, 13 kinds of aliphatic amine (C₂–C₁₆) derivatives can be separated within 40 min by reversed-phase HPLC with gradient elution. In this chromatogram, unreacted reagents peak at around 28 min, greatly decrease after fluorous solid-phase extraction, and do not interfere with the quantification of each amine. The detection limits (S/N = 3) for examined aliphatic amines are 3.6–25 fmol per 20 μL injection. We have also applied this reagent successfully to the amino acid analysis.

HPLC determination of (+)-pseudoephedrine and (-)-ephedrine in Japanese herbal medicines containing Ephedra herb using solid-phase extraction

We developed a rapid and simple HPLC method combined with solid-phase extraction (SPE) for quantitative analysis of (+)-pseudoephedrine (PEP) and (-)-ephedrine (EP) in Japanese herbal (Kampo) medicines such as Kakkon-to, Sho-seiryu-to, Goshaku-san and Bofu-tsusho-san. SPE was performed on TOYOPAK IC-SP M containing propylsulfonic groups. Determination of PEP and EP was carried out using ion-pair reversed-phase HPLC with sodium dodecyl sulfate. N-Benzyldiethylamine was used as an internal standard. The analytical procedure was validated with regard to specificity, linearity, accuracy, and precision. These data suggest that the analytical method developed in this study is useful for quantitative analysis of PEP and EP in various formulations of Kampo medicine containing Ephedra herb.     

Nano-biphasic ionic liquid systems composed of hydrophobic phosphonium salts and a hydrophilic ammonium salt

A combination of a phosphonium-type-zwitterions-lithium bis(trifluoromethanesulfonyl)imide complex and a hydrophilic ammonium salt provides a nanosegregated liquid-crystalline matrix consisting of hydrophilic ionic liquid (IL) domains and hydrophobic IL domains.