Prevention of evaporation of small-volume sample solutions for capillary electrophoresis using a mineral-oil overlay.

The suppression of evaporation of water from small volumes of sample solutions or reagents for capillary electrophoresis by the use of a mineral-oil overlay was investigated in affinophoresis applications, in which the affinity constant of a mutant protein of recombinant human galectin-1 to a lactose affinophore, a triply negative charged ion having a lactoside as an affinity ligand, was determined. When an injection was carried out from a minimum of 20 microL of an aqueous solution beneath the oil overlay, no oil contamination inside the capillary was observed, provided the capillary was cleanly cut so that the end was flat, and the polyimide coating had been removed for a distance of about 2 mm from the end. Affinophoresis was carried out using 20 microL of an affinophore solution covered with an oil overlay. The abnormalities in the electropherograms as the result of the evaporation of the water from the solution during storage prior to use in an automatic operation of a capillary electrophoresis instrument were suppressed, with respect to the formation of a base line gap, an increase in the detection time of a marker ion and an increase in the initial current. A solution in a vial could be used repeatedly for a longer period of time when overlaid with mineral oil than in the absence of an overlay. The use of a mineral-oil overlay is a simple but very efficient technique for solving the problem of the evaporation of water from small volumes of aqueous solutions for use in capillary electrophoresis.     

Direct determination of arsenic in steel by glow discharge optical emission spectrometry with argon-helium mixed gas.

In glow discharge optical emission spectrometry, an argon-helium mixed gas plasma was investigated to improve the detection sensitivity of arsenic in steel samples. The emission line of arsenic was enhanced and the background intensity was simultaneously reduced when an Ar-He plasma was employed instead of an Ar plasma, which is effective for the sensitive determination of arsenic. The detection limits were calculated to be 0.009 mass% for a 700-V Ar plasma, 0.004 mass% for a 700-V Ar-He plasma, and 0.001 mass% for a 900-V Ar-He plasma.     

Kinetic study of the degradation of a potential local anesthetic drug in serum using the DNA-based electrochemical biosensor

DNA-drug association interaction at the DNA modified screen-printed electrode for 1-methyl-2-piperidinoethylester of 2-hexoxyphenylcarbamic acid was found leading to an accumulation of the drug within the DNA layer. A procedure for the determination of drug in blood serum matrix using the protein precipitation and voltammetric measurement of the electroactive drug with the DNA biosensor was obtained and an effort was done to apply it for an assay of the drug enzymatic degradation in human and rabbit sera at 37 degrees C.     

Determination of glucose in wine and fruit juice based on a fibre-optic glucose biosensor and flow-injection analysis

A flow-injection system for the determination of glucose in various samples using a glucose biosensor based on an oxygen optrode with immobilized glucose oxidase (GOD) is described. The consumption of oxygen was determined via dynamic quenching of the fluorescence of an indicator by molecular oxygen. GOD was adsorbed on a sheet of carbon black and cross-linked with glutaraldehyde. Carbon black was used as an optical isolation to protect the optrode from interference from ambient light and sample fluorescence. The system is linear for 0.1–500 mM glucose, with an r.s.d. of 2% at 100 mM (5 measurements). The application of the system to glucose in wine and fruit juice is demonstrated. It was possible to analyse up to 60 samples per hour. The enzyme optrode was stable for more than 400 h in continuous use and was easily maintained.     

The determination of carbon,phosphorus and sulphur in steel and cast iron with a glow-discharge emission source and an atomic fluorimeter as spectral line isolator

The atomic fluorimeter, an instrument using an atomic vapour generated by cathodic sputtering for isolating resonance spectral lines emitted by an emission source, was used for detecting the vacuum ultraviolet emission lines of carbon, phosphorous and sulphur. A glow-discharge lamp was used as emission source. Detection limits of 0.012, 0.004 and 0.002% were obtained for carbon, phosphorus and sulphur respectively in cast iron and steel. A narrow-band interference filter through which the matrix emission was measured directly by a photomultiplier tube, was used in an effort to correct for varying sputtering rates from different kinds of samples. This monitoring channel proved to be efficient for the determination of phosphorus and carbon in high alloy steels, but was not necessary for sulphur since in this case working curves of the different types of steels coincided.     

Miniaturization of capillary electrochromatography using an ultrashort capillary column

The instrumentation for miniaturization of capillary electrochromatography was devised and an injection method for this apparatus was proposed. By using an ultra short capillary column (15 mm packed length, 36 mm total length, 75 microm inner diameter, packed with cation exchange supports), the separation of five biochemical compounds was performed within 1 min. The high separation efficiency of 9780 plates was achieved by using an ultrashort capillary column. The miniaturized instrumentation for capillary electrochromatography might be utilized as one of the possible methods in microfabricated analysis or in an alternative approach to it.     

Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.     

Click chemistry with O-dimethylpropargylcarbamate for preparation of pH-sensitive functional groups. A case study

Click chemistry has became an important tool for molecular constructs such as biopolymers. During the development of biodegradable multifunctional poly(ethylene oxide) (PEO) polymers suitable for click chemistry in water, an unexpected reaction leading to a mixture of triazole cycloadducts was observed. This result was attributed to an intramolecular ligand effect, and alternative conditions were evaluated. An efficient method was then implemented allowing the access in high yields to the expected triazolylcarbamate. pH sensitivity of the obtained isopropyltriazolylcarbamate was demonstrated at acidic pH.     

Microfabricated electroosmotic pump for capillary-based sequential injection analysis

A microfabricated electroosmotic pump with an integrated serpentine isolation channel was developed on a glass chip and applied to a capillary-based sequential injection analysis (SIA) system for an enzyme inhibition assay. The pump chip contains an anode reservoir, an ion-exchange membrane electric field decoupler (EFD) that also serves as a cathode reservoir, parallel pump channels and an isolation channel. A two-step etching process was adopted to etch the pump channels to a depth of 20 μm and the isolation channel to a depth of 90 μm. The pump flow rate was very stable: the relative standard deviation (RSD) of the pump rate was 1.9% for propulsion and 2.3% for aspiration. The pump chip was successfully applied to a capillary-based sequential injection analysis system with a confocal fluorescence detector. For repetitive analysis of a 13 μM fluorescein solution, an RSD of 0.6% was attained, which demonstrated the flow stability of the EOF pump. The system was then applied to an enzyme inhibition assay, the diethylenetriaminepentaacetic acid (DTPA) inhibition of the β-galactosidase-catalyzed hydrolysis of fluorescein di(β-d-galactopyranoside). Reproducible results (RSD<3.0%) were obtained.     

Determination of relative response factors of impurities in paclitaxel with high performance liquid chromatography equipped with ultraviolet and charged aerosol detectors

A case study was conducted to determine the relative response factors (RRFs) of paclitaxel-related impurities by high performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) detector and charged aerosol detector (CAD) in tandem. The peak response using CAD was independent of analyte structure in an isocratic analysis for this application. After a sample containing known and unknown impurities was analyzed with HPLC-UV-CAD, an empirical approach was developed to calculate the RRFs for all impurities. The RRFs of known impurities were also determined by linear calibration curves. For known impurities, the RRFs values determined with two approaches are comparable. The new approach is effective yet simpler to determine the RRFs for unknown impurities or degradation products since the need for obtaining authentic pure materials was eliminated.     

Formal Synthesis of Sarain A: Intramolecular Cycloaddition of an Eight‐Membered Cyclic Nitrone to Construct the 2‐Azabicyclo[3.3.1]nonane Framework

An enantioselective route to the tetracyclic skeleton of sarain A has been developed. Asymmetric reduction of an ynone introduced a chiral center which was transferred to the contiguous tertiary stereogenic centers through an Ireland–Claisen rearrangement. The 2‐azabicyclo[3.3.1]nonane framework was constructed by an unprecedented intramolecular cycloaddition of an eight‐membered cyclic nitrone. Using the steric bias of the bicyclic system, the quaternary carbon atom was constructed by a stereoselective aldol reaction. Further ring formations were performed by ring‐closing metathesis for the 13‐membered ring and an iodoamidation reaction for the pyrrolidine ring. The present synthesis has successfully provided an alternative route to the late‐stage intermediate of Overman’s synthesis.     

Photostabilized chlorophyll a in mesoporous silica: adsorption properties and photoreduction activity of chlorophyll a

Chlorophyll a was adsorbed to mesoporous silica (FSM, folded-sheet mesoporous material) to form a chlorophyll-FSM conjugate, in which a nanometer-scale interaction between chlorophyll a molecules resembles a living plant leaf. The mesopores of FSM acted as nanoscale spaces not only for an interaction between chlorophyll molecules and the silica support but also for a nanoscale interaction between the absorbed chlorophyll molecules. These interactions contribute to photostability. An increase in the amount of chlorophyll adsorbed to the pores of FSM leads to an enhancement of the photostability accompanied by a shift in the absorbance maximum to a longer wavelength. The physiological function of the chlorophyll-FSM conjugate was explored as chlorophyll-FSM exhibited the photoinduced ability to catalyze the reduction of methyl viologen (an electron carrier). The evolution of hydrogen gas was observed for 14 h without deterioration when an aqueous suspension containing chlorophyll-FSM, methyl viologen, 2-mercaptoethanol (an electron donor), and platinum was illuminated with visible light.     

Evaluation of Near Infrared Dyes as Labels for Immunoassays Utilizing Laser Diode Detection: Development of Near Infrared Dye Immunoassay (NIRDIA)

Abstract

Many recent studies of near infrared (NIR) dyes have shown their usefulness as probes for many analytical applications. Since these dyes have absorption maxima between 600 and 1000 nm, a region of low interference by most bioorganic molecules, their use as a label for biomolecules is practical. We report here the first study to evaluate the use of near infrared dyes as a quantitative label for immunoassays. A near infrared dye was derivatized with an isothiocyanate functional group and conjugated to goat anti-human immunoglobulins (GAHG). After purification by G-25 size exclusion chromatography, the conjugate was used in an immunoassay to detect and quantitate human immunoglobulins (Ig), the antigen for GAHG. Polystyrene microtiter plate wells were coated with varying amounts of human Ig. The Ig-coated wells were then exposed to an excess of NIRD-GAHG, and were subsequently read and quantitated by laser diode spectroscopy. An identical immunoassay was performed which utilized an enzyme(peroxidase)-labeIed GAHG for comparison. From these studies, the effectiveness of the NIR dye-conjugate as an accurate and sensitive quantitative probe for immunoassays was evaluated.     

RNA separation by in-capillary denaturing polymer electrophoresis with 1,2,5-thiadiazole as an additive

1,2,5-Thiadiazole improved RNA separation with in-capillary denaturing polymer electrophoresis. 1,2,5-Thiadiazole was synthesized as an extraction solvent substituted for a halogenated solvent. While 1,2,5-thiadiazole was an excellent extraction solvent and an environmentally friendly solvent, we found that 1,2,5-thiadiazole was a strong hydrophobic compound for RNA and the RNA separation performance by in-capillary denaturing polymer electrophoresis was dramatically improved. We suggest "in-capillary denaturing polymer electrophoresis" as an RNA separation that realizes the denaturing and separation simultaneously. RNA separation by the method required a strong denaturant, acetic acid, to cleave the intramolecular hydrogen. The running buffer containing acetic acid was of high conductivity and low pH, in which the condition introduced Joule heating and low sensitivity. While conventional denaturants, formaldehyde and urea, maintained small electric conductivity and neutral pH, these denaturants were too weak to achieve the RNA separation by in-capillary denaturing polymer electrophoresis. 1,2,5-Thiadiazole being a neutral molecule, both conductivity and buffer pH were able to be adjusted to a desirable strength for RNA separation. In this paper, we report that RNA separation by in-capillary denaturing polymer electrophoresis in neutral pH was achieved and the sensitivity for RNA separation was higher than that for RNA separation by in-capillary denaturing polymer electrophoresis with acetic acid.     

Synthetic studies aimed at (-)-cochleamycin A. Evaluation of late-stage macrocyclization alternatives

An efficient route to the fully functionalized ABC ring systems of the unnatural enantiomer of cochleamycin A was developed. L-(-)-Malic and L-(-)-ascorbic acids served well as starting materials for the two building blocks used to construct an (E,Z,E)-1,6,8-nonatriene intermediate. The AB part structure was assembled by way of a stereocontrolled intramolecular Diels-Alder cycloaddition via adoption of an endo transition state. From this vantage point, two general pathways were subsequently explored as to their suitability for elaboration of the CD rings. Initially examined was a protocol involving 10-membered carbocycle construction. When this approach was demonstrated not to be workable, attention was directed to 10-membered macrolactonization as an alternative tactic. Although assembly of the C-ring in this manner was easily achieved, ultimate closure of the six-membered ring to form ring D remains an unsolved problem.     

Application of sweeping to micellar electrokinetic chromatography-atmospheric pressure chemical ionization-mass spectrometric analysis of environmental pollutants.

An application study of sweeping, an on-line sample concentration technique, to micellar electrokinetic chromatography (MEKC) directly combined with mass spectrometry (MS) using an atmospheric pressure chemical ionization (APCI) interface, namely MEKC-APCI-MS, was investigated to enhance the concentration sensitivity for the analysis of environmental pollutants. Under a neutral condition, around 100-fold increase in the concentration sensitivity was achieved for several aromatic amines and alkyl phthalates as test samples by sweeping-MEKC-APCI-MS compared to conventional MEKC-APCI-MS, whereas under an acidic condition, 100 to 600-fold sensitivity enhancement was gained for similar solutes. Linearity of the corrected peak area obtained in the mass chromatogram against the sample concentration was examined for 3,4-dichloroaniline and diisopropyl phthalate (DIPP). The estimated limits of detection for 3,4-dichloroaniline and DIPP were 0.6 and 0.4 ppm, respectively, in terms of the injected sample concentration.     

Miniaturized on-line proteolysis-capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase

In this study, methodology was developed for on-line and miniaturized enzymatic digestion with liquid chromatographic (LC) separation and mass spectrometric (MS) detection. A packed capillary LC-MS system was combined with on-line trypsin cleavage of a model protein, lactate dehydrogenase, to provide an efficient system for peptide mapping. The protein was injected onto an enzymatic capillary reactor and the resulting peptides were efficiently trapped on a capillary trapping column. Different trapping columns were evaluated to achieve a high binding capacity for the peptides generated in the enzyme reactor. The peptides were further eluted from the pre-column and separated on an analytical capillary column by a buffer more suitable for the following an electrospray ionisation (ESI) MS process. An important aspect of the on-line approach was the desalting of peptides performed in the trapping column to avoid detrimental signal suppression in the ESI process. The developed on-line system was finally compared to a classical digestion in solution, with reference to peptide sequence coverage and sensitivity. It was shown that the on-line system gave more than 100% higher peptide sequence coverage than traditional digestion methods.     

Separation and determination of aluminium by single-column ion-chromatography

A quick, reliable method for the determination of Al(III) in the presence of other metal ions is presented. A Chromatographic system consisting of a low-capacity cation-exchange column, an eluent of diprotonated p-phenylenediamine, and a conductivity detector was used to measure the retention times for various cations. During the course of this work, it was found that Al(III) was eluted later than most bivalent metal ions but earlier than other tervalent metal ions. Therefore the concentration of eluent was adjusted so that an early sharp peak was obtained for Al(III) and the bivalent metal ions were eluted as a group. Through analysis of an NBS standard, as well as of solutions containing Al(III) and other metal ions, the method was shown to be precise, accurate and rapid for determination of Al(III) without interference from common bivalent metal ions.     

Limonene as an agro-chemical building block for the synthesis and extraction of bioactive compounds

The objective of this study was to transform limonene as an agro-chemical platform for the production of a wide range of added-value compounds for pharmaceutical, cosmetic and food ingredients. This molecule was also evaluated as an alternative solvent for the extraction of several bioactive compounds compared to n-hexane. Limonene was extracted from the essential oils of orange peels through a solvent-free microwave extraction technique. Limonene was successfully transformed into products with industrial interest by catalytic oxidation using three different iron catalysts. The ability of limonene to be used as an alternative solvent was performed using two simulation tools, Hansen solubility parameters (HSPs) and the Conductor-like Screening Model for Real Solvents (COSMO-RS), and via experimentation. The results indicated that limonene could be a promising green solvent and synthon for petroleum substitution in the extraction or synthesis of bioactive compounds.     

透射电镜像转角的测定及应用

论述了完全标定透射电镜像转角所需拍摄的二次曝光电子显微像的数量,推导了由已测的像转角间接计算其它像转角的一般公式,以Philips Cm200电镜为实例,说明了测定像转角的基本步骤,并以实例说明怎样利用像转角,把衍射花样的晶体学信息传递给电子显微像。