Simultaneous determination of trace of loxacin, ciprofloxacin, and sparfloxacin by micelle TLC-fluorimetry

The method of simultaneous determination of ofloxacin (OFLX), ciproflxacin (CPLX), and sparfloxacin (SPLX) by thin-layer chromatography is established, with micelle solutions as mobile phases. It is found that the optimum molar ratio of sodium dodecyl sulfate (SDS) to ethylene diamine tetraacetic acid is 0.01:0.1. On the polyamide thin-layer sheet, OFLX, CPLX, and SPLX are separated from each other, and the corresponding Rf values are 0.72, 0.55, and 0.32, respectively. The fluorescence spots are scanned with a spectrodensitometer at the excitation wavelength of 282 nm. The cut-off filter is set at 400 nm. The detection limits are 2 x 10(-6) mol/L for OFLX, 1.5 x 10(-6) mol/L for CPLX, and 1.6 x 10(-6) mol/L for SPLX, and the respective linear ranges correspondingly fell in the concentration of 1 x 10(-5) to 4 x 10(-4) mol/L for OFLX, 1 x 10(-5) to 4.5 x 10(-4) mol/L for CPLX, and 1 x 10(-5) to 4.2 x 10(-4) mol/L for SPLX. For all the three components, the relative standard deviations are in the range of 1.12-5.82%, and the recoveries are found to be 96.7-104.2% in urine and serum samples.     

Synthesis and characterization of an amphiphilic cyclodextrin, a micelle with two recognition sites

A cyclodextrin derivative (Mod-CD) was synthesized through the monoesterification of beta-cyclodextrin (beta-CD) with 3-((E)-dec-2-enyl)-dihydrofuran-2,5-dione. The compound is an interesting surfactant that can form large aggregates not only through the interaction of the hydrophobic tails as in common amphiphilic compounds but also through the inclusion of the alkenyl chain into the cavity of another Mod-CD molecule. The self-inclusion of the chain in the cavity of cyclodextrin as well as the intermolecular inclusion was demonstrated by 1H NMR measurements that were able to detect methyl groups in three different environments. Besides, in the aggregates of Mod-CD, the cavity is available to interact with external guests such as phenolphthalein, 1-amino adamantane, and Prodan. Phenolphthalein has the same binding constant with Mod-CD and beta-CD, but the equilibrium constant for the interaction with Prodan is about 2 times larger for Mod-CD than for beta-CD. The latter result is attributed to the fact that this probe interacts with the micelle in two binding sites: the cavity of the cyclodextrin and the apolar heart of the micelle as evidenced by the spectrofluorimetric behavior of Prodan in solutions containing different concentrations of Mod-CD.     

Rapid isoelectric trapping in a micropreparative-scale multicompartment electrolyzer

A multicompartmental electrolyzer called membrane-separated wells for isoelectric focusing and trapping (MSWIFT) has been developed and tested for micropreparative-scale isoelectric trapping separations. In the MSWIFT, the length of the heat conduction path from the center of the compartments to the wall is less than 1 mm. The compartments are made from 99.8% nonporous alumina that has a high heat conductivity and a high specific heat capacity, leading to adequate Joule heat dissipation even at power loads as high as 5 W. The length of any compartment parallel to the electric field (the intermembrane distance) can be selected to be multiples of 1.5 mm, leading to compartment volumes that are multiples of about 60 muL. A maximum of 20 (1.5 mm long) separation compartments can be readily assembled in the current version of MSWIFT. The MSWIFT has been used to desalt samples, isolate small ampholytic components (amino acids, peptides, and dyes), prefractionate complex protein mixtures and enrich minor components; these separations were achieved in 20-60 min.     

Simultaneous presence of two charges or two spins in a linear polyene

Summary In this work we examine the ease of finding simultaneously two charges or two spins in various AO positions of a linear neutral polyene such as octatetraene. By applying the general multi-electron population analysis on a correlated wave function we calculate contributions from various ionic and covalent (in a probabilistic sense) resonance structures as well as electron-pair distributions with parallel and anti-parallel spins; the initial wave function used is a PPP + (quasi-total) CI wave function. Based on the second and fourth order anticommutation relations (and independently of the level of the MO wave function used) we show existing relationships between the quantities considered, as well as their constant behaviour. Covalent structures depend strongly on the parity and distance between the AO positions, while ionic ones arequasi-constant along the polyene. It is also shown that when the AO positions considered are non-vicinal, (+) and (–) charges appearmutually independent and the -electron conjugation has, rather surprisingly, no effect on them.     

Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing

A new protein fractionation technique based on off-gel isoelectric focusing (IEF) is presented, where the proteins are separated according to their isoelectric point (pI) in a multiwell device with the advantage to be directly recovered in solution for further analysis. The protein fractions obtained with this technique have then been characterized with polymer nanoelectrospray for mass spectrometry (MS) analyses or with Bioanalyzer for mass identification. This methodology shows the possibility of developing alternatives to the classical two-dimensional (2-D) gel electrophoresis. One species numerical simulation of the electric field distribution during off-gel separation is also presented in order to demonstrate the principle of the purification. Experiments with pI protein markers have been carried out in order to highlight the kinetics and the efficiency of the technique. Moreover, the resolution of the fractionation was shown to be 0.1 pH unit for the separation of beta-lactoglobulin A and B. In addition, the isoelectric fractionation of an Escherichia coli extract was performed in standard solubilization buffer to demonstrate the performances of the technique, notably for proteomics applications.     

Simultaneous determination of nine endogenous steroids in human urine by polymeric-mixed micelle capillary electrophoresis

A new CE system based on the use of polymeric-mixed micelles (cholic acid, SDS and the poloxamine Tetronic(?) 1107) was developed for the simultaneous determination of nine steroids in human urine. This method allows the baseline separation and quantitation of cortisol, androstenedione, estriol, dehydroepiandrosterone sulfate, testosterone, dehydroepiandrosterone, estrone, progesterone and estradiol in less than 25 min showing to be sensitive enough to detect low concentrations of these steroids in urine samples (5-45 ng/mL). The optimized electrophoretic conditions were performed using a 50 cm × 75 μm capillary, 18 kV, 25°C, with 44 mM cholic acid, 10 mM SDS, 0.05% w/v tetronic(?) 1107, 2.5% v/v methanol, 2.5% v/v tetrahydrofuran in 5 mM borate - 5 mM phosphate buffer (pH=8.0) as a background electrolyte and a dual 210/254 UV-detection. The method can simultaneously determine 0.1-120 μg/mL, which corresponds to 5-6000 ng/mL of steroids in 2 mL urine. The recoveries ranged between 82.4 and 101.5%. Due to its simplicity, speed, accuracy and reliability, the proposed method could be a potential alternative to the traditional methodologies used with clinical purposes.     

Gel-free IEF in a membrane-sealed multicompartment cell for proteome prefractionation

A minidevice for performing gel-free proteome prefractionation via conventional IEF in soluble carrier ampholyte buffers is reported here. It consists of a compact block of polyoxymethylene in which eight samples and two electrode chambers are machined. Each of the eight sample chambers can be filled with up to 120 microL of sample and has the following size: 7 mm width, 3 mm depth and 10 mm height. The anodic and cathodic compartments have the same width and height as the sample chambers, but with a depth of 6 mm, thus accepting up to 250 microL of electrodic solutions. Focusing is in general accomplished in 2 h with a voltage gradient of up to 1000 V (7 cm electrode distance). Easy fractionation and collection of the content of the eight chambers is achieved by simply pressing a rubber diaphragm against the edges of the thin walls separating each well, this automatically breaking liquid continuity. The performance of this device has been tested by subfractionating total cell lysates of a human cancer cell line (U2Os) and of Escherichia coli bacterial cells, and by analysing the content of each chamber by mono-dimensional SDS-PAGE and 2-D maps.     

Autofocusing and ESI-MS analysis of protein digests in a miniaturized multicompartment electrolyzer

Free-solution IEF of protein digests was studied in a newly introduced MicroRotofortrade mark multicompartment electrolyzer. The fractionation was performed in a cylindrical separation chamber divided into ten compartments with or without the addition of carrier ampholytes. In the case of autofocusing mode of operation, the tryptic digest itself served as the mixture of ampholytes leading to the separation of the peptides with well-defined pI's. The focusing process was monitored visually using colored pI markers. The resulting fractions from both modes of the separation were analyzed by CE and electrospray-TOF mass spectrometer using electrospray tips microfabricated in polyimide. Additional experiments, aiming at visualization of the mass flux within the focusing compartments were performed using isotachophoretic migration of color cationic tracers. The study considered the autofocusing of both the peptides with well-defined narrow pI's as well as those showing negligible net charge in a broader pH range. Although not all peptides in the protein digests have well-defined pI's the autofocusing process can preseparate many of them leading to higher S/N in the ESI-MS signals and improved protein sequence coverage.     

Simultaneous protein tagging in two colors

The fluorescent tagging of proteins in the natural environment of the cell is an emerging technique in cell biology. In this issue of Chemistry & Biology, Gautier et al. introduce a fluorescent labeling procedure orthogonal to existing ones, enabling tagging of two different proteins in living cells.     

Simultaneous determination of 16 estrogens,dehydroepiandrosterone and their glucuronide and sulfate conjugates in serum using sodium cholate micelle capillary electrophoresis

The simultaneous determination of 16 estrogens, dehydroepiandrosterone (DHEA) and their glucuronide and sulfate conjugates by micellar electrokinetic chromatography (MEKC) with sodium cholate micelle is reported. Sodium cholate, sodium dodecylsulfate (SDS) and alpha-, beta-, gamma-cyclodextrins were studied as micelle reagents in the pH range of 7.0-10.0. Estrogens, DHEA and their glucuronide and sulfate conjugates were separated using a 50 cm x 50 microm capillary with 10 mM borate-phosphate buffer (pH 8.0) containing 50 mM sodium cholate as carrier. The method could simultaneously determine 1.0-1000 microg/mL of steroids and metabolites in 100 microL of serum by photometric detection at 214 nm within 14 min and 80 ng/mL steroids could be determined by using 2.0 mL of serum. The relative standards deviations were 6.7-7.7% at 10 microg/mL in serum. The recoveries were 89.1-92.0% with 10 microg/mL serum samples.     

Bioisosteric hybrids of two anti-inflammatory agents, rutaecarpine and piroxicam

Bioisosteric replacements were accomplished by building the structural elements of piroxicam, an anti-inflammatory drug, into the pentacyclic system of rutaecarpine, a quinazolinocarboline alkaloid, in order to modify activity and selectivity on COX-isoenzymes. The pentacyclic compounds were synthesized efficiently by employing 1-oxo-9,10,11,12-tetrahydro-4H-pyrido[1,2-b]1,2-benzothiazine 5,5-dioxide as a key intermediate, and prepared by alternative routes. Condensation of the tricyclic ketone with arylhydrazines and subsequent Fischer-indolization provided the first representatives of new heterocyclic ring systems 3 and 4.     

Ultrafast fluorescence resonance energy transfer in a micelle

Ultrafast fluorescence resonance energy transfer (FRET) from coumarin 153 (C153) to rhodamine 6G (R6G) is studied in a neutral PEO(20)-PPO(70)-PEO(20) triblock copolymer (P123) micelle and an anionic micelle (sodium dodecyl sulfate, SDS) using a femtosecond up-conversion setup. Time constants of FRET were determined from the rise time of the acceptor emission. It is shown that a micelle increases efficiency of FRET by holding the donor and the acceptor at a close distance (intramicellar FRET) and also by tuning the donor and acceptor energies. It is demonstrated that in the P123 micelle, intramicellar FRET (i.e., donor and acceptor in same micelle) occurs in 1.2 and 24 ps. In SDS micelle, there are two ultrafast components (0.7 and 13 ps) corresponding to intramicellar FRET. The role of diffusion is found to be minor in the ultrafast components of FRET. We also detected a much longer component (1000 ps) for intramicellar FRET in the larger P123 micelle.     

Simultaneous detection of two triplets: a time-resolved resonance Raman study

Solvents are known to affect the triplet state structure and reactivity. In this paper, we have employed time-resolved resonance Raman (TR3) spectroscopy to understand solvent-induced subtle structural changes in the lowest excited triplet state of thioxanthone. Density functional theory (DFT) combined with the self-consistent reaction field (SCRF) implicit solvation model has been used to calculate the vibrational frequencies in the solvents. Here, we report a unique observation of the coexistence of two triplets, which has been substantiated by the probe wavelength-dependent Raman experiments. The coexistence of two triplets has been further supported by photoreduction experiments carried out at various temperatures.     

Properties of a drag reducing micelle system

Rheological properties of a drag-reducing surfactant were investigated with the intention to find out the influence of shear rate and the duration of shear straining on the Shear Induced Structure. The ability of the surfactant to restore the broken drag-reducing network after the decrease of shear stress is well known. This property of reversible change caused by high shear at different flow conditions was compared to a more intensive mechanical straining by means of ultrasound. Observations using electronmicroscope and spectrometer are also presented.     

Separation of proteins in a multicompartment electrolyzer with chambers defined by a bed of gel beads

Multicompartment electrolyzers (MEs) with isoelectric membranes were introduced in 1989 for purifying proteins in an electric field. At the basis of ME technology there are membranes consisting of cross-linked copolymers of acrylamide and acrylamido monomers bearing protolytic groups. The technology employed for casting the membranes is an extension of the isoelectric focusing in immobilized pH gradient technique for which specific acrylamido monomers, known with the trade name of Immobiline, have been developed. However, the use of continuous membranes presents several disadvantages. Due to the mechanical characteristics of polyacrylamide, the gel must physically adhere onto a rigid support, which prevents it from collapsing. The support must have a highly porous structure in order to be permeable to proteins. The mechanical fragility of the membranes is one of the main problems that hinders the industrial scale application of ME separators. In order to overcome this problem, we propose to substitute the continuous membranes with a bed of gel beads of identical comonomer composition, obtained by an inverse emulsion polymerization process.     

Application of a multivariate approach for analyte focusing by micelle collapse‐micellar electrokinetic chromatography for analyzing sunscreen agents in cosmetics

The operating parameters that affect the performance of the online preconcentration technique “analyte focusing by micelle collapse‐MEKC (AFMC‐MEKC)” were examined using a multivariate approach involving experimental design to determine the sunscreen agents in cosmetics. Compared to the single‐variable approach, the advantage of the multivariate approach was that many factors could be investigated simultaneously to obtain the best separation condition. A fractional factorial design was used to identify the fewest significant factors in the central composite design (cCD). The cCD was adopted for evaluating the location of the minimum or maximum response in this study. The influences of the experimental variables on the response were investigated by applying a chromatographic exponential function. The optimized condition and the relationship between the experimental variables were acquired using the JMP software. The ANOVA analysis indicated that the Tris pH value, SDS concentration, and ethanol percentage influenced the separation quality and significantly contributed to the model. The optimized condition of the running buffer was 10 mM Tris buffer (pH 9.5) containing 60 mM SDS, 7 mM γ‐CD, and 20% v/v ethanol. The sample was prepared in 100 mM Tris buffer (pH 9.0) containing 7.5 mM SDS and 20% v/v ethanol. The SDS concentration in the sample matrix was slightly greater than the CMC value that makes the micelle be easily collapsed and the analytes be accumulated in the capillary. In addition, sunscreen agents in cosmetics after 1000‐fold dilution were successfully determined by AFMC‐MEKC.     

pH-controlled, polymer-mediated assembly of polymer micelle nanoparticles

We describe pH-controlled, polymer-mediated assembly of polymer micelles in aqueous media based on reversible complexation between the micelles of pyrene-labeled poly(epsilon-caprolactone)-b-poly(carboxylic acid) copolymers and proton-accepting water-soluble polymers such as poly(ethylene glycol) (PEG), poly(2-ethyl-2-oxazoline) (PEtOz), and poly(1-vinylpyrrolidone) (PVP). The key factor determining assembly phenomena was identified as the modulation of hydrogen-bonding interaction between ionizable anionic micellar shells and the proton-accepting polymers by the pH control. As pH decreased from 7.4 to 2.0, the mixture of the polymer micelles and polymers underwent assembly and formed solid hybrids at specific pH values. The micelles assembled in the hybrid could be reversibly dispersed as micelles above specific pH ranges. The assembly/disassembly behavior as well as phase transitions of the micelle/proton-accepting polymer could be precisely controlled by adjusting pH. This assembling behavior depended on the rationally designed parameters such as the chemical structure and length of micellar shell-forming poly(carboxylic acid)s and the class of proton-accepting polymers.     

Preparative-scale isoelectric focusing separation of enantiomers using a multicompartment electrolyzer with isoelectric membranes

The IsoPrime multicompartment electrolyzer, equipped with a series of isoelectric membranes with closely spaced pI values, was used for the first time for the preparative-scale separation of the enantiomers of dansyl phenylalanine with hydroxypropyl beta-cyclodextrin as resolving agent. The final separation conditions could be established easily in three successive experiments by rationally narrowing the pH steps between the neighboring isoelectric membranes. The final separation yielded products with an enantiomeric excess greater than 99.9%, at production rates of about 0.1 mg/h. The greatest experimental difficulty was caused by the relatively high salt content of the hydroxypropyl beta-cyclodextrin used, which resulted in high conductivity and limited the maximum field strength one could use.     

A reversible tube-to-rod transition in a block copolymer micelle

A remarkable morphology transition occurs with a change in temperature for a diblock copolymer [poly(ferrocenyldimethylsilane-b-dimethylsiloxane) (PFS40-b-PDMS480, PDI = 1.01)] in n-decane solution. This polymer, which forms nanotubes at 25 degrees C, rearranges to form short dense rods when the solution is heated to 50 degrees C. When the solution is cooled to 25 degrees C, the system evolves back to nanotubes. These experiments demonstrate that both structures are dynamic and represent equilibrium states of the material. Contrast matching static light-scattering measurements on the short dense rods show that the insoluble PFS core is rigid and has a length distribution similar to that seen in electron microscopy images.