The extractive technique for protein purification based on two-phase separation in aqueous micellar solutions (aqueous micellar two-phase system (AMTPS)) is reviewed. The micellar solution of a nonionic surfactant, such as polyoxyethyl-ene alkyl ether, which is most frequently used for protein extraction, separates into two phases upon heating above its cloud point. The two phases consist of a surfactant-depleted phase (aqueous phase) and a surfactant-rich phase. Hydrophilic proteins are partitioned to the aqueous phase and hydrophobic membrane proteins are extracted into the sur-factant-rich phase. Because of the methodological simplicity and rapidity, this technique has become an effective means, and thus has been widely used for the purification and characterization of proteins. In contrast to polyoxyethylene alkyl ether, micellar solutions of a zwitterionic surfactant, such as alkylammoniopropyl sulfate, separate below the critical temperature. Alkylglucosides can also separate into two phases upon adding water-soluble polymers. Recently, these two-phase systems have been exploited for protein separation. Additionally, hydrophobic affinity ligands, charged polymers, and ionic surfactants have been successfully used for controlling the extractability of proteins in AMTPS.
The importance of vitamin C to the human body is widely acknowledged throughout the globe. The deficiency of this vitamin leads to various diseases. In view of its importance, numerous methods including spectrophotometric ones have been developed for its determination in pharmaceuticals, foods and biological samples. A comprehensive review of the available spectophotometric methods for the determination of ascorbic acid is presented.
The thermodynamics of the inclusion reaction of flavonol, 7-methoxyflavonol and 4-phenyl-7-hydroxycoumarin (L) with β-cyclodextrin (CD) has been studied fluorometrically with special reference to the effect of organic solvents (S). The equilibrium constants for this reaction were determined in aqueous methanol, ethanol, acetonitrile, acetone, dimethyl sulfoxide, and N,N-dimethylformamide. The dependence of the constants on the content of the respective S was attributable to the substitution of S included in CD with L as CD·nS+L⇔CD·L+nS. Also, the inclusion constants of S with CD, βn=[CD·nS][CD]-1[S]-n, were found to be linearly correlated with the solubility parameter of S for both n=1 and 2. When aqueous methanol was employed as a mobile phase, the retention behavior of coumarins on a reversed-phase HPLC column was studied in the presence of β-CD as a mobile-phase component. The change in the capacity factors could be reasonably explained by the same inclusion scheme as that mentioned above.
Strontium Kα X-ray fluorescence intensity of SrTiO3 (001) single crystal is measured as a function of azimuthal and polar angles of detection. Anisotropy is observed due to the interference effect of fluorescent X-rays in the solid. X-Ray fluorescence holography is discussed from the viewpoint of chemical analysis. X-Ray photoelectron intensity anisotropy is compared with the present measurement.
The performance of polyester film was examined as a disposable cover of the sample-carrier (silicon wafer). The cover offers convenience and economy for the total reflection X-ray fluorescence (TXRF) analysis of solution samples. Moreover, the measurement of Al Kα (1.487 keV) and P Kα (2.013 keV) peaks is possible because of the reduction of Si Kα X-rays (1.740 keV) from the sample-carrier. Not only the trace elements (Cr, Mn, Co, Ni and Cu) but also the low sensitivity elements (Al and P) in JSS iron and steel standard samples were successfully analyzed.
The two-photon excited fluorescence of fluorescein and Rhodamine B in a flowing sample cell was observed using a self mode-locked Ti-sapphire laser (180 fs pulse). The detection limit of fluorescein was less than 2 molecules in the probe volume. The single molecule detection of Rhodamine was successfully performed in a flowing cell, and the number of photon bursts agreed approximately with that expected.
An automated gas chromatograph/electron capture detector (GC/ECD) system is developed for the monitoring of major seven halocarbons in the atmosphere (CCl2F2: CFC-12, CCl3F: CFC-11, CCl2FCClF2: CFC-113, CH3CCl3, CCl4, CHCl=CCl2, and CCl2=CCl2) at the tropospheric background concentration levels. By using two columns in series for each of two groups divided by the volatility of the compounds, the system requires neither cryogen for preconcentration nor any physical/chemical dryer for water removing. A sample amount of ca.18 ml STP is sufficient to detect these species, and analysis of sub-ambient atmospheric-pressure sample in a canister is also possible. Monitoring of these halo-carbon concentrations was carried out at 90-min intervals in the urban area.
A method for the separation and determination of amino acids by capillary electrophoresis with laser-induced fluores-cence detection has been developed. At first, the condition for the derivatization of amino acids with fluorescein isothio-cyanate (FITC) was studied; it was proved to be optimal at 50°C for 5 h. Next, the separation of 20 FITC-amino acids was attempted by using an unmodified fused silica capillary. Actually, 18 kinds of FITC-amino acids were separated with 96 mM sodium borate buffer (pH 9.5) containing either 25% acetone (separation system 1) or 10% methanol (separation system 2). The resolution of 19 FITC-amino acids was attained with 80 mM sodium borate buffer (pH 9.2) containing 45 mM α-cyclodextrin (separation system 3). Finally, we succeeded in separating 20 FITC-amino acids by combining two migration systems (2 and 3). The FITC-amino acids were detected by argon-ion laser-induced fluorescence (Ex 488 nm, Em 520 nm). The detection limits of the amino acids obtained with separation systems 1 and 2 were 0.052-1.01 fmol.
A negatively charged metal chelate, bis(bathocuproinedisulfonic acid)-copper(I), was formed and stably solubilized in an aqueous solution of a water-soluble polymer, poly(N-isopropylacrylamide) (PNIPAAm), at room temperature. When the solution was warmed at 40°C, PNIPAAm became water-insoluble. It then formed a gum-like aggregate (polymer phase) having a very small volume. The chelate was difficult to be extracted to the polymer phase in the absence of counter ions, but was efficiently incorporated when appropriate amounts of quaternary ammonium ion are present as the counter ions. The recovery of the chelate increased with increasing the concentration or the hydrophobic properties of the counter ions, indicating the ion-pair extraction of the charged chelate into the polymer phase. In the presence of 1 mM dodecyltrimethylammonium chloride, the recovery of the chelate was quantitative. After the polymer phase was dissolved by 1-propanol in the hole of a 96-hole microplate, the absorbance at 450 nm of copper(I) chelate was measured. A slightly curved calibration was obtained in the copper(II) concentration range from 5×10-9 M to 5×10-7 M. The standard deviation was 6% (n=5) for 5×10-8 M copper(II) ions.
Some anion-exchange resins modified with water-soluble metalloporphyrins exhibited an ability to catalyze simultane-ously both uricase- and catalase-like reactions. The resin modified with manganese-tetrakis(4-sulfophenyl)porphine (Mn-TPPSr), which showed the highest activity among the resins tested, was useful for the indirect determination of uric acid by measuring the amount of formaldehyde produced. The calibration curve was linear over the range from 10 μg to 200 μg of uric acid with a relative standard deviation of 1.8% (n=10) for 200 μg uric acid. Mn-TPPSr was also applied for the determination of uric acid in sera.
A simple and mild on-line photo-oxidative decomposition method for the highly sensitive flow-injection spectrophotometric determination of total phosphorus in river and waste waters was developed for the first time by using low pressure mercury lamps. Organic and inorganic phosphorus compounds in sample solutions were injected into a carrier solution, which merged with the decomposition solution containing sulfuric acid and potassium peroxodisulfate. The solution components were decomposed oxidatively to orthophosphate while flowing in a photo-reactor. After the decomposition, orthophosphate was determined spectrophotometrically based on the formation of Molybdenum Blue. The photo-reactor consisted of two low pressure mercury lamps, a poly(terafluoroethylene) (PTFE) tubing and a reflection mirror. The PTFE tubing was wound directly around the mercury lamps, which were covered with reflection mirror. The detection limit for phosphorus was about 0.001 mg l-1, and the sample throuphput was about 20 samples per hour for total phosphorus determination. By using the photo-reactor, most of the phosphorus compounds were decomposed completely, whereas some condensed phosphates were less susceptible to photo-oxidation. The analytical results for practical samples obtained by the proposed method showed a good correlation with those obtained by using a heat-oxidation method, which indicates that the content of the condensed phosphates difficult to be decomposed was very scarce in the practical samples.
An on-line sensor was developed by using a micro-machining technique for the continuous measurement of extracellular glutamate. The sensor was made by bonding two glass plates together. One of the plates has a rectangular flow channel which is connected to both a sampling and an outlet capillary. The other has a carbon film-based electrochemical cell, which consists of an enzyme-modified electrode surrounded by polymer film to form a thin-layer-channel flow cell. The sensor has a high analyte conversion efficiency due to the cell structure. As a result, we obtained a low detection limit of 44 nM (S/N=3). The cathodic current started to increase about 10 s after sample introduction at a flow rate of 2 μl/min due to its small inner volume. Using the sensor, we can monitor the extracellular L-glutamate increase caused by stimulating it with KCl and γ-aminobutyric acid (GABA) by locating the sampling capillary very close to the cell.
An ion-selective piezoelectric sensor (ISP) was developed for the rapid determination of benzydamine hydrochloride in serum and urine. The ISP device was fabricated by coating a PVC membrane containing benzydamine phosphotungstate on one electrode of a thickness-shear mode piezoelectric quartz crystal. The selective adsorption of benzydamine ion across the membrane caused a decrease in the oscillating frequency; also the logarithm of the frequency shift has a linear relation to the logarithm of the concentration of benzydamine over the ranges 2×10-7 - 8×10-3 M with a detection limit of 2.0×10-7 M at pH 7.0. Influencing factors were investigated and optimized. The results for real samples obtained by the proposed method were in agreement with those by the conventional non-aqueous titrimetric method.
A titanium dioxide electrode using copper(II) ion was developed for measuring glycine. The copper(II)-glycine complex interacted on the titanium dioxide electrode surface. Use of the copper redox system enabled indirect measurement of glycine with good reproducibility without elution of the titanium ion. The calibration curve was determined and the effects of temperature, pH, the scanning rate, concomitant compounds, and the lifetime of the electrode were examined. Under optimal conditions, the current response was linear in the concentration range of 0.02 - 0.10 mM glycine.
A square-root Kalman filter combined with spectrometry has been applied to determining the concentrations of vitamin B1, vitamin B2, vitamin B6 and nicotinamide in synthetic mixtures and in a commercial composite vitamin B tablet. A simultaneous assay of the components in synthetic mixtures containing the above mentioned four B-complex vitamins was successfully achieved with satisfactory recoveries of 97.0 - 103.3%. The components in a commercial composite vitamin B tablet were determined simultaneously, and the obtained results are in good agreement with the labeled contents of the tablet.
The chemiluminescence (CL) of Ru(phen)32+ was applied to HPLC determination of oxalic acid, which was separated by a C18 reverse-phase column with a mobile phase of 0.02 mol/l NH4Ac, mixed with 0.25 mol/l Ru(phen)32+ and 2.0 mmol/l Ce(SO4)2 (0.08 mol/l H2SO4), and then passed through a homemade CL detector. The reaction of Ce(IV) oxidized Ru(phen)32+ and the oxalic acid emitted light. The detection limit was 6.2×10-6 mol/l for oxalic acid at a S/N ratio of 3, the relative standard deviation for 5 replicate injections of 1×10-3 mol/l oxalic acid standard was calculated as 5.6%, and the lin-ear calibration range was 1×10-5 to 4×10-3 mol/l. The method was successfully applied to determination of oxalic acid in tea.
An ion-pair high-performance liquid chromatography with chemiluminescence detection using tris(2,2′-bipyridyl)ruthe-nium(II) [Ru(bpy)32+] was developed for the determination of yohimbine (YOH) in human serum. A sample solution, containing narceine as an internal standard, was treated with a Bond-Elut Certify disposable column, and the purified fraction was separated on a TSKgel ODS-80Ts column. The recovery of YOH at 50 ng/ml was 97.2±4.4% (mean±SD, n=5). The detection limit was calculated to be 30 ng/ml of YOH in serum (S/N=3). The method was successfully applied to drug monitoring of the YOH level in the serum of healthy volunteers.
A high-performance liquid chromatography with chemiluminescence detection using tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)32+] for the simultaneous determination of phencyclidine and its major metabolites in rat urine was developed. The urine sample was applied onto a disposable cartridge column (Bond Elut Certify) of silica solid-phase extraction. The charged sample was then washed with an acidic solution and methanol prior to its elution with 2% ammonium hydroxide in methanol, and the eluate was chromatographed. In this method, a TSKgel ODS 80Ts column and a mobile phase containing 60% methanol, 50 mM phosphate buffer (pH 7.0) and 0.12 mM tris(2,2′- bipyridyl)chloride hexahydrate in 10 mM sulfuric acid as a reagent solution were used. The calibration curves for phencyclidine and its metabolites showed good linearity over the range 25 - 400 ng ml-1 per 20 μl injection. As a result, the method was successfully applied to a simultaneous determination of phencyclidine and its three major metabolites in actual rat urine.
The mercury (5 - 100 ng of Hg2+ and/or CH3Hg+) in drinking water (25 ml) containing 2,3-dimercaptopropane-1-sulfonate (DMPS, 15 µg) and sodium acetate buffer (1 mmol, pH=6.0) was concentrated on two Sep-Pak C18 cartridges in series. The mercury-DMPS complex was then eluted with methanol and adjusted to 2.00 ml. A portion (50 µl) was introduced into a graphite cuvette and was atomized according to a temperature program. The method detection limit (3 σ) was 0.053 μg/l; the calibration graphs were linear up to 4.00 μg/l. Accuracies of 97.4 - 99.3% were obtained when testing a synthetic natural water standard (Standard Reference Material 1641c). The proposed method could be applied to the determination of methylmercury in air with a detection limit of 1.3 ng.