RESUMEN
A silica-based chiral monolithic column prepared by sol-gel process and chemical modification of chiral selector was used for enantioseparation of dansyl amino acids and hydroxy acids by capillary electrochromatography (CEC) and mu-high-performance liquid chromatography (mu-HPLC). L-Prolinamide was modified as a chiral selector. The chiral stationary phase (CSP), the chiral complex of Cu(II) with L-prolinamide, provides an anodic electroosmotic flow (EOF) in CEC. The EOF was found to be dependent on applied electric field strength, the pH, and the composition of mobile phases. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pore. D-Enantiomers migrated before L-enantiomers except for dansyl-(Dns)-DL-Ser. The separation efficiencies of up to 17600 (D) and 13,200 plates m(-1) (L) were achieved for the separation of DL-indole-3-lactic acid.
Asunto(s)
Aminoácidos/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Cromatografía/métodos , Compuestos de Dansilo/aislamiento & purificación , Hidroxiácidos/aislamiento & purificación , Prolina/análogos & derivados , Prolina/química , Dióxido de Silicio , Acetonitrilos , Tampones (Química) , Cobre/química , Concentración de Iones de Hidrógeno , Indoles/aislamiento & purificación , Microscopía Electrónica de Rastreo , EstereoisomerismoRESUMEN
A blue-light-emitting diode was incorporated as a fluorescence-excitation light source into a polyester microchannel chip fabricated by in situ polymerization. Placing the light-emitting facet of the diode close to the microchannel obviated any need for an additional optical arrangement. Fluorescence from the sample was transmitted by an optical fiber incorporated into the microchip perpendicular to the LED. FITC labeled amino acids were separated in the presence of 5 mM SDS by using the microchip and were detected by LED-induced fluorescence.
Asunto(s)
Aminoácidos/aislamiento & purificación , Microquímica/métodos , Aminoácidos/análisis , Electroforesis/instrumentación , Electroforesis/métodos , Diseño de Equipo , Tecnología de Fibra Óptica , Fluorescencia , Fibras Ópticas , Fenilalanina/análisis , Fenilalanina/aislamiento & purificación , Poliésteres , Sensibilidad y Especificidad , Valina/análisis , Valina/aislamiento & purificaciónRESUMEN
At least three transport systems function in targeting nuclear-encoded chloroplast proteins to the chloroplast thylakoid membrane. One of these systems requires a thylakoid pH gradient and is named the DeltapH-dependent protein transport system. A similar DeltapH export system of Escherichia coli contains four components, twin arginine translocation A (TatA), TatB, TatC, and TatE. TatC is a major component of the DeltapH-dependent protein transporter in E. coli and functions in the translocation of tightly folded proteins across membranes. We have isolated four transposon-inserted albino mutants named albino and pale green 2 (apg2) from Arabidopsis thaliana and showed that the transposons were inserted into different sites of a single gene. The APG2 gene product (named cpTatC) has sequence similarity with bacterial TatC and contains six putative transmembrane domains, including bacterial TatC proteins and a transit peptide in its N terminus. apg2 mutants showed albino phenotypes and could not grow in soil. The apg2 plastids were highly vacuolated, lacked internal membrane structures and lamellae of the thylakoid membrane, and contained many densely stained globule structures, like undifferentiated proplastids. Immunoblot analysis detected no thylakoid membrane proteins such as D1, light-harvesting complex, and OE23 in apg2 plastids, whereas soluble proteins such as rubisco large and small subunits were not decreased. These results indicate an essential role of cpTatC in chloroplast development, especially in thylakoid membrane formation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Secuencia de Bases , Transporte Biológico Activo , Proteínas Portadoras/genética , Proteínas de Cloroplastos , Cloroplastos/metabolismo , Elementos Transponibles de ADN/genética , ADN de Plantas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes de Plantas , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
18-Crown-6-tetracarboxylic acid (18C6H4) is a chiral selector with high selectivity for the enantioseparation of solutes bearing the primary amine function. This work presents the simultaneous separation of positional enantiomers of methyl-DL-tryptophans by using 18C6H4 as an additive to the background electrolyte. Separation conditions such as pH, the concentration of 18C6H4, and the applied voltages have critical inference on the simultaneous separation. The addition of cyclodextrins as anionic surfactants to the background electrolyte did not improve the separation. The selector-selectand interactions between 18C6H4 and the positional enantiomers have been investigated. It was observed that both the position and type of substituents contribute to the enantioselectivity. The migration order and resolution depended on the distance from the substituents to the asymmetric carbon of the enantiomers.
Asunto(s)
Éteres Corona , Electroforesis Capilar/métodos , Éteres Cíclicos , Triptófano/análogos & derivados , Ciclodextrinas , Isomerismo , Metilación , Dodecil Sulfato de Sodio , Estereoisomerismo , Triptófano/química , Triptófano/aislamiento & purificaciónRESUMEN
Simultaneous separation of 12 o-, m- and p-positional enantiomers of tyrosine and fluorophenylalanine has been achieved by the use of optically active 18-crown-6-tetracarboxylic acid (18C6H4) as an additive in the background electrolyte. It has been investigated that the separation conditions such as pH, electroosmotic flow, the concentration of 18C6H4 and applied voltages have critical influence on the simultaneous separation. Based on the information of electropherograms obtained, the interaction between the chiral selector and positional enantiomers has been discussed. It was shown that alpha-methyl group in the amino acid enantiomers make the resolution be reduced, and that o-, m- and p-substituents have an influence on the resolution.
Asunto(s)
Éteres Corona , Electroforesis Capilar/métodos , Éteres Cíclicos/química , Fenilalanina/aislamiento & purificación , Concentración de Iones de Hidrógeno , Fenilalanina/químicaRESUMEN
A new type of chiral monolithic column was successfully developed for the enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography (LE-CEC) in this work. The monolithic column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and the chiral selector L-phenylalaninamide. After being conditioned with Cu(II) aqueous solution, the ligand exchange-chiral stationary phase (LE-CSP) possesses positive charges. When the external electric field was applied in CEC, electroosmotic flow (EOF) was generated on the surface of LE-CSP in the direction from the cathode to the anode. The EOF was found to be dependent on the applied electric field strength and the composition of the mobile phase. With the increase of pH of the mobile phase, the EOF showed a tendency to decrease. Scanning electron microscopy showed that the chiral monolithic column has a continuous skeleton and large through-pore structure. The separation efficiency (theoretic plate numbers) for the separation of Dns-DL-Leu reached up to 9.0 x 10(4) plates m(-1) for the D-enantiomer and 6.6 x 10(4) plates m(-1) for the L-enantiomer, by using pH 5.5, acetonitrile/0.50 mM Cu(Ac)2-50 mM NH4Ac (7:3) as mobile phase. The reproducibility and lifetime were satisfactory. CEC was carried out with conventional capillary electrophoresis equipment without pressurizing the ends of the capillary. No bubble was formed during the operation, after degassing the mobile phase and conditioning the column.
Asunto(s)
Aminoácidos/aislamiento & purificación , Cromatografía Capilar Electrocinética Micelar/métodos , Compuestos de Dansilo/aislamiento & purificación , Fenilalanina/análogos & derivados , Fenilalanina/química , Dióxido de Silicio/química , EstereoisomerismoRESUMEN
Enantiometers of D,L-phenylalanine were separated by capillary electrophoresis based on the principle of ligand exchange. Copper (II) complex of 4-hydroxy-L-proline was used as chiral selector. The separation and the migration order of D- and L-phenylalanine were strongly affected by adding an anion surfactant sodium dodecylsulfate (SDS). Without SDS in the electrolyte, the separation was also carried out but the resolution was very small. With SDS added into the electrolyte, the resolution decreased with increasing concentration of SDS until 5.0 mM. When the concentration of SDS in the electrolyte was over 5.0 mM, inversion of the migration order of DL-phenylalanine was observed and the resolution was also increased with increasing concentration up to 20 mM. It was interesting to find that the inversion of the migration order took place not only in the enantioscparation but also in the positional isomers. A family of a fluorinated amino acid, o-, m- and p-fluoro-D,L-phenylalanine was separated and the inversion of the migration order is discussed.
Asunto(s)
Cromatografía Capilar Electrocinética Micelar/métodos , Fenilalanina/aislamiento & purificación , Estereoisomerismo , Fenilalanina/químicaRESUMEN
A continuous preconcentration system for nitrate ions was developed using cation exchange tubing made from Nafion perfluorosulfonic acid membrane. This method is based on ion exclusion effects and reverse osmosis phenomena. The system was evaluated by connecting it to an ion chromatograph. The concentration ratios could be increased by raising the pressures between the two sides of the cation exchange tubing. Twenty-fold concentration of nitrate ion was achieved when the pump pressure was 20 x 10(5) Pa. The relative standard deviations of the preconcentration ratio at four different pump pressures, 5, 10, 15 and 20 x 10(5) Pa were 1.2-2.8% (n = 5).
RESUMEN
Inexpensive and disposable polyester microchips were fabricated through photolithographic and wet-chemical etching procedure, followed by replication using an imprinting method at room temperature. Laboratory-scale laser-induced fluorescence equipment was employed as a detection system. The generation of electroosmotic flow (EOF) on the polyester channels was discussed in this paper. Surfactants in the running buffer had a significant effect on the EOF depending on their types. The epsilon potential of the electric double layer formed by adsorbing sodium lauryl sulfate molecules on the wall of polyester channels seemed to be constant within the buffer pH investigated. EOF could also be suppressed to zero by adding polyoxyethylene 23 lauryl ether into the running buffer. The separation of two laser dyes was obtained using polyester chips through both micellar electrokinetic chromatography and capillary zone electrophoresis. The polyester channels modified with 10-undecen-1-ol exhibited a dramatically high-separation efficiency compared with the conventional fused-silica capillary tubes.
Asunto(s)
Electroforesis Capilar/métodos , Poliésteres/química , Concentración de Iones de Hidrógeno , ÓsmosisRESUMEN
A divalent cation-selective electrode, which utilizes a lipophilic resin as a matrix for the sensing membrane, and which has long-term stability has been developed. The sensing membrane is a lipophilic acrylate resin which is impregnated with a solution of 1-decylalcohol and the calcium salt of bis[4-(1,1,3,3-tetramethylbutyl) phenyl] phosphate at concentrations of 0.08 g ml(-1) each. The electrode exhibited nearly equal selectivity to Ca(2+) and Mg(2+) ions and could be used as a water hardness sensor. The electrode shows a Nernstian response with a slope of 29 mV decade(-1) to both Ca(2+) and Mg(2+) ions in the concentration range from 10(-5) M to 10(-1) M and could be used in the pH range from 3 to 10 for the determination of 10(-3) M Ca(2+) and Mg(2+) solutions. The initial performance of the electrode could be maintained for 1 year, since the lifetime test of the electrode was conducted in tapwater at a continuous flow rate of 4 ml min(-1). The hardnesses of tapwater and upland soil extracts were determined using the developed electrode and the analytical results were in good agreement with those obtained by chelatometric titration using an EDTA solution as the titrant. A coefficient factor of correlation 0.998 was obtained between the electrode method and titrimetry. The long-term stability of the electrode was found to be due to strong affinity of 1-decylalcohol to the lipophilic acrylate resin.
RESUMEN
An azobenzene-modified gamma-cyclodextrin stationary phase (Az gamma-CDSP) was prepared and its photo- and temperature-responses for the retention of perylene and pentacene were investigated using a mixture of methanol and water as the mobile phase in micro-HPLC. The retention of perylene slightly increased, whereas that of pentacene significantly decreased by UV light irradiation to Az gamma-CDSP. These retentions recovered upon irradiation with visible light. Both retentions decreased upon an increase in the column temperature. It was presumed that the trans-azobenzene moiety acts as a preventive cap for perylene and a spacing for pentacene in filling the CD cavity. An azobenzene-modified stationary phase changed its retention behavior with the column temperature and the light irradiation. An improvement in the micro-HPLC system and the optimization of the molecular structure of the photo-responsive stationary phase would provide selective retention control by the irradiation of light in micro-separation systems.
Asunto(s)
Compuestos Azo/química , Ciclodextrinas/química , Compuestos Azo/efectos de la radiación , Cromatografía Líquida de Alta Presión , Ciclodextrinas/efectos de la radiación , Cinética , Fotoquímica , Temperatura , Rayos UltravioletaRESUMEN
Thermal lens spectroscopy was combined with total internal reflectance spectroscopy to develop a novel, highly sensitive analytical method that can detect nonfluorescent as well as fluorescent analytes at surfaces and interfaces. It was verified that when the total internal reflection method is coupled with thermal lens spectroscopy (TIR-TLS), the thermal lens effect is induced by only the evanescent wave. The ability of depth profiling was shown. The detection limit of TIR-TLS was at an absorbance of 3.0 x 10(-5) unit for Sudan II acetone solution, which is better than that of attenuated total reflection by a factor of hundreds. In addition, the adsorption of acridine orange on silanol groups on a glass surface could be monitored directly by TIR-TLS, and the adsorption isotherm agreed well with Langmuir's model. The dependence of surface density of anionic silanol groups on pH was determined by TIR-TLS measurements of aqueous acridine orange solutions buffered in the pH range between 2.7 and 11.5.
RESUMEN
The transcription factor VP1 regulates maturation and dormancy in plant seeds by activating genes responsive to the stress hormone abscisic acid (ABA). Although activation involves ABA-responsive elements (ABREs), VP1 itself does not specifically bind ABREs. Instead, we have identified and cloned a basic region leucine zipper (bZIP) factor, TRAB1, that interacts with both VP1 and ABREs. Transcription from a chimeric promoter with GAL4-binding sites was ABA-inducible if cells expressed a GAL4 DNA-binding domain::TRAB1 fusion protein. Results indicate that TRAB1 is a true trans-acting factor involved in ABA-regulated transcription and reveal a molecular mechanism for the VP1-dependent, ABA-inducible transcription that controls maturation and dormancy in plant embryos.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Unión al ADN/metabolismo , Leucina Zippers , Oryza/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Oryza/fisiología , Proteínas de Plantas , Unión Proteica , Elementos de Respuesta , Semillas/genética , Semillas/fisiología , Homología de Secuencia de Aminoácido , Transducción de Señal , Distribución Tisular , Transactivadores , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
ACGT-containing ABA response elements (ABREs) have been functionally identified in the promoters of various genes. In addition, single copies of ABRE have been found to require a cis-acting, coupling element to achieve ABA induction. A coupling element 3 (CE3) sequence, originally identified as such in the barley HVA1 promoter, is found approximately 30 bp downstream of motif A (ACGT-containing ABRE) in the promoter of the Osem gene. The relationship between these two elements was further defined by linker-scan analyses of a 55 bp fragment of the Osem promoter, which is sufficient for ABA-responsiveness and VP1 activation. The analyses revealed that both motif A and CE3 sequence were required not only for ABA-responsiveness but also for VP1 activation. Since the sequences of motif A and CE3 were found to be similar, motif-exchange experiments were carried out. The experiments demonstrated that motif A and CE3 were interchangeable by each other with respect to both ABA and VP1 regulation. In addition, both sequences were shown to be recognized by a VP1-interacting, ABA-responsive bZIP factor TRAB1. These results indicate that ACGT-containing ABREs and CE3 are functionally equivalent cis-acting elements. Furthermore, TRAB1 was shown to bind two other non-ACGT ABREs. Based on these results, all these ABREs including CE3 are proposed to be categorized into a single class of cis-acting elements.
Asunto(s)
Ácido Abscísico/farmacología , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Elementos de Respuesta , Factores de Transcripción/metabolismo , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Unión Proteica , TransactivadoresRESUMEN
An injection method for a large amount of headspace gas which enables determination of trace amounts of very volatile organic compounds (VVOCs), dichlorodifluoromethane, chloromethane, vinyl chloride, bromomethane, chloroethane and trichlorofluoromethane in all kinds of environmental water was developed. A gas phase equilibrated with the water phase in a vial was purged with helium for a short time. The VVOCs were then introduced into a trapping tube packed with Tenax TA, which had been cooled using carbon dioxide. After trapping, the VVOCs were thermally desorbed and put into a GC-MS system for subsequent analysis. This method is applicable to various types of samples.
Asunto(s)
Hidrocarburos/análisis , Contaminantes Químicos del Agua/análisis , Cromatografía de Gases , Indicadores y Reactivos , Japón , Temperatura , Abastecimiento de Agua/análisisRESUMEN
An analytical gas chromatography-mass spectrometry (GC-MS) method using negative ion chemical ionization (NICI) has been investigated for the determination of trace tributyltin (TBT) and triphenyltin (TPhT) compounds in sea water. TBT and TPhT were extracted from samples as chloride under the acidic condition of HCl. Doping of the GC system with a dilute HBr-methanolic solution resulted in direct detection of the chlorides of TBT, TPhT and tripentyltin (TPenT, internal standard). As the result of HBr doping, a sharp peak of the respective organotin bromides appeared: during GC analysis, halogen exchange from the chloride to the bromide occurred. NICI-MS was highly selective and sensitive for the detection of TBT. TPhT and TPenT bromides. In the selected ion monitoring mode of NICI-MS, the minimum detectable amounts defined as the signal equal to three times the standard deviation (3 sigma) of the baseline noise were 20 and 25 pg ml-1 for TBT and TPhT, respectively. These amounts are approximately 250-400 times better than those in electron impact mode. The combination of GC using an apolar capillary column doped with a dilute HBr-methanolic solution and NICI-MS made it possible to determine TBT and TPhT at less than the ng l-1 level in sea water.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Compuestos Orgánicos de Estaño/análisis , Agua de Mar/química , Compuestos de Trialquiltina/análisis , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
To measure the difference between samples in situ, two types of open-ended photoacoustic cells were constructed. One type was a differential open-ended photoacoustic cell with a differential microphone, which connected two equivalent open-ended cell chambers through two waveguides. The other design used single open-ended photoacoustic cell chambers, each with an electric condenser microphone. A pair of these was used to suppress background signal by subtraction of the signals. The performance of these cells was compared to that of a conventional photoacoustic apparatus with a chopper. Detection limits of Sudan red for those cells were about 1.29 and 2.35 ng for a differential open-ended cell and a single open-ended cell, respectively. The photoacoustic cells were then applied to the pulse mode operation, using a nitrogen laser as a pulse light source. The laser beams were led to the photoacoustic cells through quartz optical fibers. Using this instrument setup, depth profiling analysis was carried out for two-layer samples made of poly(methyl methacrylate) (PMMA) or polystyrene (PS) films. A linear relationship was obtained between the signal delay time and the film thickness. The delay time of the signal (τ) was expressed in terms of the thermal diffusivity (κ/cm(2) s(-)(1)) of the film as τ (ms) â (2.25 × 10(-3)) x (µm) κ(-1/2). The method was successful in measuring the thickness of transparent surface layers. We applied this technique to observe the generation of a certain chromophore, thought to be melanin, under the surface of human skin tanned by exposure to long-wavelength UV light.
RESUMEN
L-Aromatic amino acid imprinted polymers were prepared using azobisnitriles as either photoinitiators or thermal initiators at temperature ranging from 4 to 60 degrees C. Methacrylic acid (MAA) was used as functional monomer and ethylene glycol dimethacrylate (EDMA) was used as cross-linker. The result polymers were ground and sieved to particles < or = 10 microns, filled into the capillary columns and used for enantiomeric separations in capillary electrochromatographic (CEC) mode. The polymer particles < or = 25 were packed into high performance liquid chromatographic (HPLC) columns and used for enantioseparations in the HPLC mode. The mobility and chiral separation of some amino acids were studied on CEC and HPLC columns at different temperature. The relationships of separation factor and column temperatures and demonstrated in linear between logarithm of the separation factor (in alpha) and the inverse of absolute temperature (1/T). Some thermodynamic parameters, such as the apparent change of enthalpy (delta H zero) and the apparent change of entropy (delta S zero), were obtained using van't Hoff plots. The information obtained from the thermodynamic study is discussed in developing strategies for chiral recognitions of amino acids by molecular imprinting technique.
Asunto(s)
Aminoácidos/aislamiento & purificación , Cromatografía/métodos , Electroforesis Capilar , Polímeros/química , Estereoisomerismo , Temperatura , Aminoácidos/química , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión/métodos , Reactivos de Enlaces Cruzados , Metacrilatos , Fenilalanina/química , Fenilalanina/aislamiento & purificación , TermodinámicaRESUMEN
The use of molecularly imprinted polymer polymerized in capillary for the separation of amino acid enantiomers by electrochromatography is described. The substrate-selective polymers were prepared by using L-phenylalanine anilide as print molecule and methacrylic acid and/or 2-vinylpyridine as the functional monomers, which is believed to interact both ionically and through hydrogen bonding with the print molecule. Several aspects of the polymer preparation were investigated, including the treatment of the inside surface of the capillary, the composition of the polymers and the running conditions of the capillary electrochromatography. Such separation was highly specific and depended on the presence of both the print molecule and the functional monomer in the polymerization mixture. This preliminary report demonstrates a novel and simple method for the development of the capillary electrochromatographic separation of amino acid enantiomers using molecularly imprinted polymer.
Asunto(s)
Aminoácidos/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Electroforesis Capilar/métodos , Polímeros , Reactivos de Enlaces Cruzados , Estructura Molecular , Fenilalanina/análogos & derivados , EstereoisomerismoRESUMEN
Fourteen impacted teeth in thirteen patients aged 9 to 17 years were either repositioned or transplanted, followed by postoperative orthodontic treatment. Successful results were achieved in twelve of the teeth. In one tooth, ankylosis of the root prevented a successful outcome, and deficient bone healing of the alveolar process led to an unsuccessful result in a second tooth.