Short Separation Time Research Articles

SEPARATION AND PURIFICATION OF THREE FLAVONOIDS FROM DAPHNE GENKWA SIEB. ET ZUCC.: COMPARISON IN PERFORMANCE BETWEEN MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY AND HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

The two methods of medium-pressure liquid chromatography (MPLC) and high-speed countercurrent chromatography (HSCCC) are compared in the separation and purification of crude extract from Daphne genkwa Sieb. et Zucc. The MPLC method was performed with the mobile phase composed of methanol-0.1% aqueous acetic acid in the gradient elution which yielded apigenin, 3′-hydroxygenkwanin, and genkwanin at high purities of 95.3%, 94.0%, and 91.5%, respectively, for 230 min. The same three components were isolated by HSCCC also at high purities of 93.4%, 94.3%, and 92.8%, respectively, using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:7:5:5, v/v) for 400 min. Although both methods produced effective separations of Daphne genkwa Sieb. et Zucc., MPLC gave much shorter separation time, whereas HSCCC yielded substantially greater amounts of target compounds due to avoiding irreversible sample loss onto the solid support with less consumption of the solvent. The chemical structures of purified three compounds were identified by HPLC, ESI-MS, and 1H NMR.

Modulation of dendritic synaptic processing in the lateral superior olive by hyperpolarization-activated currents

We have previously shown that mice lateral superior olive (LSO) neurons exhibit a large hyperpolarization-activated current (I(h) ), and that hyperpolarization-activated cyclic-nucleotide-gated type 1 channels are present in both the soma and dendrites of these cells. Here we show that the dendritic I(h) in LSO neurons modulates the integration of multiple synaptic inputs. We tested the LSO neuron's ability to integrate synaptic inputs by evoking excitatory post-synaptic potentials (EPSPs) in conjunction with brief depolarizing current pulses (to simulate a second excitatory input) at different time delays. We compared LSO neurons with the native I(h) present in both the soma and dendrites (control) with LSO neurons without I(h) (blocked with ZD7288) and with LSO neurons with I(h) only present peri-somatically (ZD7288+ computer-simulated I(h) using a dynamic clamp). LSO neurons without I(h) had a wider time window for firing in response to inputs with short time separations. Simulated somatic I(h) (dynamic clamp) could not reverse this effect. Blocking I(h) also increased the summation of EPSPs elicited at both proximal and distal dendritic regions, and dramatically altered the integration of EPSPs and inhibitory post-synaptic potentials. The addition of simulated peri-somatic I(h) could not abolish a ZD7288-induced increase of responsiveness to widely separated excitatory inputs. Using a compartmental LSO model, we show that dendritic I(h) can reduce EPSP integration by locally decreasing the input resistance. Our results suggest a significant role for dendritic I(h) in LSO neurons, where the activation/deactivation of I(h) can alter the LSO response to synaptic inputs.

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry for analysis of Dechloranes

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry (LC/APPI-MS/MS) was investigated as an instrumental method for the analysis of the halogenated norbornene flame retardants, Mirex, Dechloranes 602, 603, 604, and Dechlorane Plus (DP). The LC separation was optimized by screening a variety of stationary and mobile phases, resulting in a short LC separation time of 5 min. Different atmospheric pressure ionization approaches were examined including electrospray ionization, atmospheric pressure chemical ionization, and APPI, each with and without post-column addition. APPI without post-column addition was chosen for providing the best ionization response. The optimized LC/APPI-MS/MS approach resulted in instrument detection limits ranging between 25 and 50 pg. Good linearity was also achieved (up to 25.0 ng/µL; R >0.999). The method was applied to extracts of environmental samples including surface water, fish and sediments for screening purposes, and the results agreed well with those obtained by gas chromatography/mass spectrometry.

A novel silica based click lysine anion exchanger for ion exchange chromatography

Ion chromatography (IC) is one of the most powerful analysis technologies for the determination of charged compounds. A novel click lysine stationary phase was prepared via Cu(I) catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) and applied to the analysis of inorganic ions. The chromatographic evaluation demonstrated good performance (e.g. the plate number of thiocyanate is ∼50,000 plates m(-1)) and effective separation ability for the common inorganic anions with aqueous Na(2)SO(4) eluent. The separation mechanism was observed to be mainly dominated by ion exchange interaction. The retention of these analytes is highly dependent on the pH value of eluent. Compared with the lysine stationary phase prepared via the conventional manner, the click lysine exchanger demonstrated shorter retention time and better ion separation characteristics under the same chromatographic conditions, which is a great advantage for rapid separation and analysis of inorganic ions.

Analyses of non-steroidal anti-inflammatory drugs by on-line concentration capillary electrochromatography using poly(stearyl methacrylate–divinylbenzene) monolithic columns

This study describes the ability of on-line concentration capillary electrochromatography (CEC) coupled with UV or mass spectrometry (MS) for the determination of nine common non-steroidal anti-inflammatory drugs (NSAIDs) in water samples. A series of poly(stearyl methacrylate–divinylbenzene) (poly(SMA–DVB)) monolithic columns, which were prepared by single step in situ polymerization of divinylbenzene (DVB), stearyl methacrylate (SMA) and vinylbenzenesulfonic acid (VBSA, charged monomer), were developed as separation columns for the first time. The effects of polymerization condition of monolithic columns on analyte separations were examined, and the results indicated that separation performances were markedly improved in monolithic columns prepared with short reaction time (3 h) and low SMA:DVB ratio (40/60 ratio of SMA:DVB). Subsequently, an on-line concentration step of step-gradient elution was combined to this CEC system, and by optimizing the difference in eluent strength between the sample matrix and mobile phase, all NSAIDs detection sensitivity were improved (limit of detection (LOD) was 3.4–10 μg/L for UV, and 0.01–0.19 μg/L for MS). When compared to the best CE and LC reports on NSAIDs analyses so far, this on-line concentration CEC method provided better detection ability within shorter separation time (12 min) when either UV or MS detector was employed. This is the first report for on-line concentration CEC with MS detection applied in trace solute analyses of real samples.

Study on Method of Protein Complex Detection of Lung Cancer

An experimental method of protein detection by non-gel sieving capillary electrophoresis with laser induced fluorescence(NGS-CE-LIF) was established and improved. The method was applied to the analysis of protein complex of lung cancer and compared to capillary electrophoresis with UV detection (CE-UV) and polyacrylamide gel electrophoresis(PAGE). The protein was derived by fluorescein isothiocyanate. The optimized conditions such as 0.05% (w/V) poly(ethylene oxide) (300000) as sieving medium, laboratory buffer TBE (pH 10.0) as electrophoresis buffer, separation voltage of 15 kV, temperature of 15 degrees C and argon ion laser (lambda(ex) = 488 nm, lambda(em) = 520 nm) as detector were employed. Under the conditions, the proteins under the range of 6.5 - 200 kDa were completely separated in 15 min with good separation efficiency. The average theoretical plate number (N) is 9.12 x 10(4)/m, the detection limit is 2.8 x 10(-4) g/L. The results showed that the improved method has the advantages of good separation efficiency, short separation time and the concentration of sieving medium was easy to adjust. The detection rate and efficiency of the method for the protein complex detection of lung cancer are better than those of CE-UV and PAGE.

One-pot synthesis of polyfunctionalized 4 H-pyran derivatives bearing fluorochloro pyridyl moiety

Polyfunctionalized 4 H-pyran derivatives bearing fluorochloro pyridyl moiety were readily prepared in high yields via one-pot multicomponent reaction catalyzed by piperidine. This present protocol provides an efficient synthetic route to the target compounds with the characteristics of short reaction time, high yield, and easy separation of the products.

Silica spheres coated with C18-modified gold nanoparticles for capillary LC and pressurized CEC separations

Nonporous monodispersed silica spheres of 1.3 microm were coated with gold nanoparticles (AuNPs) and subsequently coated with n-octadecanethiol. By transmission electron microscopy analysis, the average diameter of the AuNPs on the silica spheres was determined to be 12 nm. The chromatographic and electrochromatographic properties of self-assembled n-octadecanethiol AuNP-coated silica microspheres (C18-AuNPs-SiO2) were investigated using a group of nonpolar PAHs. The stationary phase appears to display a characteristic reversed-phase behavior. Higher separation efficiency and shorter separation times were obtained using pressurized CEC (pCEC) compared with capillary LC (CLC). A maximum column efficiency of about 2.5x10(5) plates per meter and less than 18 min separation time for benzene were obtained in pCEC while only 66 507 plates per meter and an analysis time of nearly 100 min were observed in CLC mode. A chemical stability test of the C18-AuNPs-SiO2 stationary phase under extremely high and low pH conditions demonstrated that it is stable at pH 12 and 1 for at least 60 h. The results confirm that C18-AuNPs-SiO2 possesses a high rigidity to withstand high packing pressures and can be used as a good stationary phase for CLC and pCEC.

Application of phase optimized liquid chromatography to oligopeptide separations

Stationary phase optimized liquid chromatography (POPLC) has been applied to the separation of oligopeptides. The retention factors and theoretical plate numbers of 13 peptides were determined on five different stationary phases. Based on these values, an optimal stationary phase composition of 250 mm total length consisting of 3 segments of 20 mm octadecyl silica, 10 mm phenyl silica and 220 mm embedded polar octadecyl silica was calculated by the optimizer software. Good agreement between the calculated and experimental chromatograms was observed. In order to achieve short analysis time and baseline separation of all peptides gradient elution and optimization of the column temperature were performed. The optimized mobile phase conditions consisted of (A) acetonitrile and (B) 0.025 M aqueous sodium phosphate buffer, pH 3.0, operated at 10% A (0–12 min) followed by 10–50% A (12–32 min) at a flow rate of 0.5 mL min −1 and column temperature of 35 °C. Using these conditions all peptides could be separated in less than 30 min with good resolution and peak symmetry.

CE separation approaches for combinations of anthracyclines and taxanes

For the first time the versatility of CE is demonstrated for the separation of different types of anticancer drugs - anthracyclines and taxanes simultaneously. The use of these drugs in combination therapy for cancer has sparked interest in the development of methods for potential application. The simultaneous analysis of anthracyclines and taxanes can significantly increase a sample throughput of a clinical laboratory. The study shows the potential of CE for such a challenge: anthracyclines and taxanes were separated by CZE, MEKC and MEEKC. The MEEKC method was successfully applied to these compounds for the first time and was characterised by very short separation time, high efficiencies of peaks and was proven to be generic for the separation of different combinations of anthracyclines and taxanes. This separation approach could be highly beneficial for clinical analysis if applied with a sensitive detection system. MEKC and high-speed MEEKC methods were proven to show good potential in their application to plasma samples.

Classical Wigner method with an effective quantum force: Application to reaction rates

We construct an effective "quantum force" to be used in the classical molecular dynamics part of the classical Wigner method when determining correlation functions. The quantum force is obtained by estimating the most important short time separation of the Feynman paths that enter into the expression for the correlation function. The evaluation of the force is then as easy as classical potential energy evaluations. The ideas are tested on three reaction rate problems. The resulting transmission coefficients are in much better agreement with accurate results than transmission coefficients from the ordinary classical Wigner method.

Tandem RF-only quadrupole mass filter with quadrupolar excitation

We report the computer simulation results of a tandem RF-only quadrupole mass filter. This mass filter consists of two short quadrupole rod sets, Q1 and Q2. The first quadrupole, Q1, is operated as a higher mass reject filter to remove lower q ions. The main filter, Q2, is a RF-only quadrupole, and it is used to give suitable narrow pass band on q axis. Quadrupole excitation by additional RF voltage leads to instability bands along q axis between q = 0 and q = 0.2, and this instability band can be used to remove low q ions with m = M 0 − ∞ mass range. Quadrupole excitation creates a narrow pass band near q 0 = 0.9080 in Q2. R 0.1 = 1150 could be achieved at short separation time n = 40 in Q2.

Differences in protein distribution between human plasma preparations, EDTA‐plasma and heparin‐plasma, analyzed by non‐denaturing micro‐2‐DE and MALDI‐MS PMF

The effects of plasma preparation methods on the status of human plasma proteins were analyzed by non-denaturing micro-2-DE followed with polypeptide assignment with MALDI-TOF MS and PMF. In order to facilitate the separation of high-molecular-mass plasma proteins (up to ca. 2x10(3) kDa) in short separation time, agarose micro-IEF gels were employed. The comparisons of the 2-DE patterns between EDTA-plasma (1.0 mg EDTA/mL blood) and heparin-plasma (0.025 mg heparin/mL blood) revealed the differences in protein distribution around pI 5.4-6.5 and apparent molecular mass of ca. 1x10(3) kDa, which were mainly attributed to the presence of the complexes of complement C4b and C4b-binding protein in heparin-plasma and their absence in EDTA-plasma. The distribution of several spots around pI 5.0-5.6 and apparent molecular mass 1.2-1.5x10(2) kDa was also found to be different; the fragments of complement C3 and C4 were detected in heparin-plasma but not in EDTA-plasma. The 2-DE pattern of high-heparin-plasma (0.50 mg heparin/mL blood) showed pI changes of three plasma proteins, fibronectin, complement factor B, and pre-alpha-inhibitor when compared with that of heparin-plasma, suggesting the interactions between heparin and the proteins. These results demonstrated that subtle changes in plasma proteins, caused by the different plasma preparation procedures, can be analyzed by non-denaturing agarose-IEF/micro-2-DE followed by MALDI-MS and PMF.

Separation of complex mixtures of fluorobenzoic acids by capillary electrophoresis

Fluorobenzoic acids are important intermediates in the synthesis of antibacterial drugs. Conditions for the separation of mixtures of twenty-five acids by CE have been optimized. A set of conditions with phosphate buffer (pH 3.1), successive multiple ionic-polymer layer (SMIL) coating capillary, and negative separation voltage provided a short time separation based on the difference in the acidic dissociation constant (pK(a)) of the sample acids. Addition of ACN to the separation solution improved the selectivity resulting in a broader distribution of migration times of the samples. The addition of tetradecyltrimethylammonium chloride below the CMC was also effective in changing the migration pattern of fluorobenzoic acids. Furthermore, practical utility was demonstrated through quantitative studies on the optimized condition including the durability of the SMIL coating.

New SPE column packing material: Retention assessment method and its application for the radionuclide chromatographic separation

The preparation of the OASIS®-HLB sorbent based solid phase extraction (SPE) resins and their application for the 177Lu radioisotope separation were investigated. Di-(2-ethylhexyl) orthophosphoric acid (HDEHP) impregnated OASIS-HLB sorbent based SPE resins (OASIS-HDEHP) were successfully developed from this investigation. The wettable porosity structure of the moderately extractant impregnated OASIS-HDEHP resins is favorable for the effective diffusion of polar and ionic solutes giving good separation performance. Its good wetting ability offers ease of packing into conventional chromatographic columns. Their off-gassing-free operation makes OASIS-HDEHP columns good for long term use with highly consistent elution dynamics (several dozens of separations perfectly achievable on the same column). The simple method for the capacity factor (k’) evaluation was developed to facilitate the characterization of the SPE chromatographic resin column. A competent procedure using OASIS-E30RS resin (one member of the OASIS-HDEHP resin group) for the separation of no-carrier added (n.c.a) 177Lu from the bulk amount of Yb target was developed. This separation procedure has showed very good performance with several prominent advantages such as the much shorter separation time (5–6 hours) and high reproducibility. Its high adsorption capacity for Yb and Lu makes it ideal for the separation of the bulky sample (50 mg Yb target for the 20 g weight resin column) for the production of the several Ci of 177Lu radioactivity.

Pressurized capillary electrochromatographic assay of trimethoprim impurities using 1 μm particle-based columns

One micrometre silica particles, derivatized with C18, were electrokinetically packed into a 75-μm-i.d. capillary. The resulting column was evaluated for the separation of trimethoprim (TMP) and its impurities using pressurized capillary electrochromatography (pCEC), starting from a capillary liquid chromatographic (CLC) separation. These samples require gradient elution when separated by high performance liquid chromatography (HPLC), but with the new columns isocratic elution suffices for their separation by CLC or pCEC. Only 70,000 theoretical plates/m for impurity C were achieved using CLC mode at relative low pressure (78 bar) although very small particles were utilized. When a voltage above 2 kV (50 V/cm) was applied, unknown peaks appeared, which was assumed due to an electrophoretic effect with the unknown peaks resolving as a result of the applied voltage. In order to minimize these unfavorable contributions, only a low voltage was applied, still leading to higher separation performances and shorter separation times than in CLC. The optimal analyzing conditions in pCEC included a pressure of 78 bar, an applied voltage of 1 kV, and a mobile phase consisting of 80 mM sodium perchlorate (pH 3.1)/methanol (60/40, v/v). These conditions were used to separate and quantify four major impurities in TMP within 22 min. The obtained calibration curves were linear ( r > 0.9980) in concentration ranges between 0.005 and 0.1 mg/mL for impurities A and C; 0.02 and 0.10 mg/mL for impurity F; and 0.01 and 0.10 mg/mL for impurity H. The detection limits (S/N = 3) for impurities A, C, F, and H were 0.52, 0.84, 3.18, and 2.41 μg/mL, respectively. The calibration curves were successfully applied to analyze spiked bulk samples, with mean recoveries ranging from 92% to 110%. The developed method can therefore be considered simple, rapid, and repeatable.

Determination of Polybromodiphenyl Ethers and Polybromobiphenyls in Electrical and Electronic Equipments by Gas Chromatography-Mass Spectrometry

A method for the determination of polybromobiphenyls (PBBs) and polybromodiphenyl ethers (PBDEs) in electrical and electronic equipments by GC-MS has been developed. The pretreatment procedures, such as solvent selection, extraction method, and column type, were discussed in detail, and standard mass spectral library of PBBs and PBDEs was established. The PBBs and PBDEs were identified by the combination of retention time, isotopic rule of bromine, characteristic ion, and retrieved reference library. The detection limit was as low as 5 mg kg −1, and the recovery rates of PBBs and PBDEs were 92.5% and 91.3%, respectively. The relative standard deviation (RSD) was lower than 4.3%. The most important characteristic of this method was its short separation time, which was within 6 min and it is the fastest that has ever been reported. Results that met the directions of Restriction of Hazardous Substances Directive (RoHS) were obtained. This method can be used to analyze the bromine-based flame retardant in electrical and electronic equipments, plastic, rubber, and decorative materials for automobiles.

Liquid–liquid extraction of aluminium(III) from mixed sulphate solutions using sodium salts of Cyanex 272 and D2EHPA

Liquid–liquid extraction of aluminium(III) from sulphate medium using sodium salt of Cyanex 272 and D2EHPA in kerosene have been studied. The influences of the following parameters were investigated: equilibrium pH, extractant concentration, organic/aqueous phase ratio, temperature and diluent type. Extraction of Al(III) increased with increasing equilibrium pH and extractant concentration. Maximum Al(III) extraction was achieved in the equilibrium pH range of 3.2–3.6 and 3.5–4.2 for Na-Cyanex 272 and Na-D2EHPA, respectively. Temperature had a positive effect on Al(III) extraction in case of Na-Cyanex 272. On the other hand, when Na-D2EHPA was used, increasing temperature had negligible effect on Al(III) extraction. Aliphatic diluents were preferred compared to aromatic diluents because of higher extraction, good separation factor, short equilibrium time and good phase separation. Quantitative Al(III) extraction efficiency was achieved with 0.30 M extractant in two stages, at an aqueous:organic (A:O) phase ratio of 1:1. Complete stripping of Al(III) from the loaded organic containing 2.4 g/L Al(III) was carried out using 1.0 M H 2SO 4 at an A:O phase ratio 1:1 in a single stage.

Fractionation of proteins with two-sided electro-ultrafiltration

Downstream processing is a major challenge in bioprocess industry due to the high complexity of bio-suspensions itself, the low concentration of the product and the stress sensitivity of the valuable target molecules. A multitude of unit operations have to be joined together to achieve an acceptable purity and concentration of the product. Since each of the unit operations leads to a certain product loss, one important aim in downstream-research is the combination of different separation principles into one unit operation. In the current work a dead-end membrane process is combined with an electrophoresis operation. In the past this concept has proven successfully for the concentration of biopolymers. The present work shows that using different ultrafiltration membranes in a two-sided electro-filter apparatus with flushed electrodes brought significant enhancement of the protein fractionation process. Due to electrophoretic effects, the filtration velocity could be kept on a very high level for a long time, furthermore, the selectivity of a binary separation process carried out exemplarily for bovine serum albumin (BSA) and lysozyme (LZ) could be greatly increased; in the current case up to a value of more than 800. Thus the new two-sided electro-ultrafiltration technique achieves both high product purity and short separation times.

Determination of volatile phenols in wine using high-performance liquid chromatography with a coulometric array detector

A new high-performance liquid chromatography method using a coulometric array detector to simultaneously analyse 4-ethylphenol, 4-ethylguaiacol, 4-vinylphenol, and 4-vinylguaiacol in wine was established. This procedure offers important advantages as it does not require sample preparation, with the exception of filtration, and performs chromatographic separation in short time, making control of wine production processes easier. The method is linear up to concentrations of 2000 μg L −1 and precise (R.S.D. < 3%). Limits of detection are low (1–3 μg L −1) and suitable for analytical requirements in the oenological field. When compared to gas-chromatography-flame ionisation detection, the proposed method gives similar results with a shorter execution time.