Microchip electrospray: improvements in spray and signal stability during gradient elution by an inverted postcolumn makeup flow.

PubMed

Jung, Stephanie; Effelsberg, Uwe; Tallarek, Ulrich

2011-12-01

Dynamic changes in mobile phase composition during high-performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray modes. We investigate the impact of the eluent composition on spray stability and MS response by infusion and injection experiments with a small tetrapeptide in water-acetonitrile mixtures. The employed HPLC/electrospray (ESI)-MS configuration uses a microchip equipped with an enrichment column, a separation column, and a makeup flow (MUF) channel. One nano pump is connected to the separation column, while a second one delivers solvent of exactly inverted composition to the MUF channel. Both solvent streams are united behind the separation column, before the ESI tip, such that the resulting electrosprayed solution always has identical composition during a gradient elution. Analyte peak parameters without and with MUF compensation are determined and discussed with respect to the electrospray mode and eluent composition. The postcolumn MUF significantly improves spray and signal stability over the entire solvent gradient, without compromising the performance of the HPLC separation column. It can also be conveniently implemented on microchip platforms.

Spatial wavefield gradient-based seismic wavefield separation

NASA Astrophysics Data System (ADS)

Van Renterghem, C.; Schmelzbach, C.; Sollberger, D.; Robertsson, J. OA

2018-03-01

Measurements of the horizontal and vertical components of particle motion combined with estimates of the spatial gradients of the seismic wavefield enable seismic data to be acquired and processed using single dedicated multicomponent stations (e.g. rotational sensors) and/or small receiver groups instead of large receiver arrays. Here, we present seismic wavefield decomposition techniques that use spatial wavefield gradient data to separate land and ocean bottom data into their upgoing/downgoing and P/S constituents. Our method is based on the elastodynamic representation theorem with the derived filters requiring local measurements of the wavefield and its spatial gradients only. We demonstrate with synthetic data and a land seismic field data example that combining translational measurements with spatial wavefield gradient estimates allows separating seismic data recorded either at the Earth's free-surface or at the sea bottom into upgoing/downgoing and P/S wavefield constituents for typical incidence angle ranges of body waves. A key finding is that the filter application only requires knowledge of the elastic properties exactly at the recording locations and is valid for a wide elastic property range.

Separation of metalloporphyrins from metallation reactions by liquid chromatography and electrophoresis.

PubMed

Duff, G A; Yeager, S A; Singhal, A K; Pestel, B C; Ressner, J M; Foster, N

1987-04-24

The analytical separation of the indium and manganese complexes of three synthetic, meso-substituted, water-soluble porphyrins from their respective free bases in metallation reaction mixtures is described. The ligands tetra-3N-methylpyridyl porphyrin, tetra-4N-methylpyridyl porphyrin and tetra-N,N,N-trimethylanilinium porphyrin are complexed with In (III) and Mn (III) and are separated from residual free base by high-performance liquid chromatography (HPLC) in acidic conditions with gradient elution on ODS bonded stationary phase. Electrophoretic separation is achieved on both cellulose polyacetate strips and polyacrylamide tube gels under basic conditions. Although analytical separations can be achieved by both HPLC and electrophoresis, only HPLC is suitable for the development of preparative scale separations. Column chromatography, ion-pairing and ion-suppression HPLC techniques fail to separate such highly charged and closely related aromatic compounds.

Development of a novel amide-silica stationary phase for the reversed-phase HPLC separation of different classes of phytohormones.

PubMed

Aral, Hayriye; Aral, Tarık; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

2013-11-15

A novel amide-bonded silica stationary phase was prepared starting from N-Boc-phenylalanine, cyclohexylamine and spherical silica gel (4 µm, 60 Å). The amide ligand was synthesised with high yield. The resulting amide bonded stationary phase was characterised by SEM, IR and elemental analysis. The resulting selector bearing a polar amide group is used for the reversed-phase chromatography separation of different classes of thirteen phytohormones (plant hormones). The chromatographic behaviours of these analytes on the amide-silica stationary phase were compared with those of RP-C18 column under same conditions. The effects of different separation conditions, such as mobile phase, pH value, flow rate and temperature, on the separation and retention behaviours of the 13 phytohormones in this system were studied. The optimum separation was achieved using reversed-phase HPLC gradient elution with an aqueous mobile phase containing pH=6.85 potassium phosphate buffer (20 mM) and acetonitrile with a 22 °C column temperature. Under these experimental conditions, the 12 phytohormones could be separated and detected at 230 or 270 nm within 26 min. Copyright © 2013 Elsevier B.V. All rights reserved.

Linear solvation energy relationships in normal phase chromatography based on gradient separations.

PubMed

Wu, Di; Lucy, Charles A

2017-09-22

Coupling the modified Soczewiñski model and one gradient run, a gradient method was developed to build a linear solvation energy relationship (LSER) for normal phase chromatography. The gradient method was tested on dinitroanilinopropyl (DNAP) and silica columns with hexane/dichloromethane (DCM) mobile phases. LSER models built based on the gradient separation agree with those derived from a series of isocratic separations. Both models have similar LSER coefficients and comparable goodness of fit, but the LSER model based on gradient separation required fewer trial and error experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

CZE separation of strawberry anthocyanins with acidic buffer and comparison with HPLC.

PubMed

Comandini, Patrizia; Blanda, Giampaolo; Cardinali, Andrea; Cerretani, Lorenzo; Bendini, Alessandra; Caboni, Maria Fiorenza

2008-10-01

Anthocyanins, the major colourants of strawberries, are polar pigments that are positively charged at low pH. Herein, we have assessed a new analytical method for the separation of anthocyanins using CZE. Acidic buffer solutions (pH <2) were employed in order to maintain pigments in the cation flavylium form and achieve high molar absorptivity at 510 nm. These spectral properties enabled us to identify strawberry anthocyanins in a preliminary stage by detection in the visible range, although the method was optimised at 280 nm to obtain the best S/N. The effects of buffer composition highlighted the necessity of adding an organic modifier to the running buffer to obtain a suitable separation. The electrophoretic method permitted the separation of the three main anthocyanins of strawberry extracts, namely pelargonidin 3-glucoside (Pg-glu), pelargonidin 3-rutinoside and cyanidin 3-glucoside. The electrophoretic results, expressed as retention time and separation efficiency of the major anthocyanin (Pg-glu), were compared to those achieved in HPLC, the analytical technique traditionally used for the investigation of anthocyanins in vegetable matrix. The content of Pg-glu in strawberries (cv. Camarosa), calculated with HPCE and HPLC methods, resulted respectively in 11.41 mg/L and 11.37 mg/L.

Gradient RP-HPLC method for the determination of potential impurities in atazanavir sulfate.

PubMed

Chitturi, Sreenivasa Rao; Somannavar, Yallappa Somappa; Peruri, Badarinadh Gupta; Nallapati, Sreenivas; Sharma, Hemant Kumar; Budidet, Shankar Reddy; Handa, Vijay Kumar; Vurimindi, Hima Bindu

2011-04-28

This paper proposes a simple and selective RP-HPLC method for the determination of process impurities and degradation products (degradants) of atazanavir sulfate (ATV) drug substance. Chromatographic separation was achieved on Ascentis(®) Express C8, (150mm×4.6mm, 2.7μm) column thermostated at 30°C under gradient elution by a binary mixture of potassium dihydrogen phosphate (pH 3.5, 0.02M) and ACN at a flow rate of 1.0ml/min. A photodiode array (PDA) detector set at 250nm was used for detection. Stress testing (forced degradation) of ATV was carried out under acidic, alkaline, oxidative, photolytic, thermal and humidity conditions. In presence of alkali, ATV transformed into cyclized products and the order of degradation reaction is determined by the method of initial rates. The unknown process impurities and alkaline degradants are isolated by preparative LC and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectral data. The developed method is validated with respect to sensitivity (lod and loq), linearity, precision, accuracy and robustness and can be implemented for routine quality control analysis and stability testing of ATV. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of temperature gradient on liquid-liquid phase separation in a polyolefin blend.

PubMed

Jiang, Hua; Dou, Nannan; Fan, Guoqiang; Yang, Zhaohui; Zhang, Xiaohua

2013-09-28

We have investigated experimentally the structure formation processes during phase separation via spinodal decomposition above and below the spinodal line in a binary polymer blend system exposed to in-plane stationary thermal gradients using phase contrast optical microscopy and temperature gradient hot stage. Below the spinodal line there is a coupling of concentration fluctuations and thermal gradient imposed by the temperature gradient hot stage. Also under the thermal gradient annealing phase-separated domains grow faster compared with the system under homogeneous temperature annealing on a zero-gradient or a conventional hot stage. We suggest that the in-plane thermal gradient accelerates phase separation through the enhancement in concentration fluctuations in the early and intermediate stages of spinodal decomposition. In a thermal gradient field, the strength of concentration fluctuation close to the critical point (above the spinodal line) is strong enough to induce phase separation even in one-phase regime of the phase diagram. In the presence of a temperature gradient the equilibrium phase diagrams are no longer valid, and the systems with an upper critical solution temperature can be quenched into phase separation by applying the stationary temperature gradient. The in-plane temperature gradient drives enhanced concentration fluctuations in a binary polymer blend system above and below the spinodal line.

Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications.

PubMed

Aral, Tarık; Aral, Hayriye; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

2015-01-01

A novel mixed-mode stationary phase was synthesised starting from N-Boc-glutamine, aniline and spherical silica gel (4 µm, 60 Å). The prepared stationary phase was characterized by IR and elemental analysis. The new stationary phase bears an embedded amide group into phenyl ring, highly polar a terminal amide group and non-polar groups (phenyl and alkyl groups). At first, this new mixed-mode stationary phase was used for HILIC separation of four nucleotides and five nucleosides. The effects of different separation conditions, such as pH value, mobile phase and temperature, on the separation process were investigated. The optimum separation for nucleotides was achieved using HILIC isocratic elution with aqueous mobile phase and acetonitrile with 20°C column temperature. Under these conditions, the four nucleotides could be separated and detected at 265 nm within 14 min. Five nucleosides were separated under HILIC isocratic elution with aqueous mobile phase containing pH=3.25 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 265 nm within 14 min. Chromatographic parameters as retention factor, selectivity, theoretical plate number and peak asymmetry factor were calculated for the effect of temperature and water content in mobile phase on the separation process. The new column was also tested for nucleotides and nucleosides mixture and six analytes were separated in 10min. The chromatographic behaviours of these polar analytes on the new mixed-model stationary phase were compared with those of HILIC columns under similar conditions. Further, phytohormones and phenolic compounds were separated in order to see influence of the new stationary phase in reverse phase conditions. Eleven plant phytohormones were separated within 13 min using RP-HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 230 or 278 nm. The best separation

Monitoring gradient profile on-line in micro- and nano-high performance liquid chromatography using conductivity detection.

PubMed

Zhang, Min; Chen, Apeng; Lu, Joann J; Cao, Chengxi; Liu, Shaorong

2016-08-19

In micro- or nano-flow high performance liquid chromatography (HPLC), flow-splitters and gradient elutions are commonly used for reverse phase HPLC separations. When a flow splitter was used at a high split-ratio (e.g., 1000:1 or higher), the actual gradient may deviate away from the programmed gradient. Sometimes, mobile phase concentrations can deviate by as much as 5%. In this work, we noticed that the conductivity (σ) of a gradient decreased with the increasing organic-solvent fraction (φ). Based on the relationship between σ and φ, a method was developed for monitoring gradient profile on-line to record any deviations in these HPLC systems. The conductivity could be measured by a traditional conductivity detector or a capacitively coupled contactless conductivity detector (C(4)D). The method was applied for assessing the performance of an electroosmotic pump (EOP) based nano-HPLC. We also observed that σ value of the gradient changed with system pressure; a=0.0175ΔP (R(2)=0.964), where a is the percentage of the conductivity increase and ΔP is the system pressure in bar. This effect was also investigated. Copyright © 2016. Published by Elsevier B.V.

Spectroscopic characterization and quantitative determination of atorvastatin calcium impurities by novel HPLC method

NASA Astrophysics Data System (ADS)

Gupta, Lokesh Kumar

2012-11-01

Seven process related impurities were identified by LC-MS in the atorvastatin calcium drug substance. These impurities were identified by LC-MS. The structure of impurities was confirmed by modern spectroscopic techniques like 1H NMR and IR and physicochemical studies conducted by using synthesized authentic reference compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. These impurities were detected by newly developed gradient, reverse phase high performance liquid chromatographic (HPLC) method. The system suitability of HPLC analysis established the validity of the separation. The analytical method was validated according to International Conference of Harmonization (ICH) with respect to specificity, precision, accuracy, linearity, robustness and stability of analytical solutions to demonstrate the power of newly developed HPLC method.

Drift and separation in collisionality gradients

DOE PAGES

Ochs, I. E.; Rax, J. M.; Gueroult, R.; ...

2017-07-20

Here we identify a single-particle drift resulting from collisional interactions with a background species, in the presence of a collisionality gradient and background net flow. We also analyze this drift in different limits, showing how it reduces to the well known impurity pinch for high-Zi impurities. We find that in the low-temperature, singly ionized limit, the magnitude of the drift becomes mass-dependent and energy-dependent. Furthermore, by solving for the resulting diffusion-advection motion, we propose a mass-separation scheme that takes advantage of this drift, and analyze the separative capability as a function of collisionally dissipated energy.

Drift and separation in collisionality gradients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ochs, I. E.; Rax, J. M.; Gueroult, R.

Here we identify a single-particle drift resulting from collisional interactions with a background species, in the presence of a collisionality gradient and background net flow. We also analyze this drift in different limits, showing how it reduces to the well known impurity pinch for high-Zi impurities. We find that in the low-temperature, singly ionized limit, the magnitude of the drift becomes mass-dependent and energy-dependent. Furthermore, by solving for the resulting diffusion-advection motion, we propose a mass-separation scheme that takes advantage of this drift, and analyze the separative capability as a function of collisionally dissipated energy.

Chiral separation of the β2-sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies.

PubMed

Ullrich, Thomas; Wesenberg, Dirk; Bleuel, Corinna; Krauss, Gerd-Joachim; Schmid, Martin G; Weiss, Michael; Gübitz, Gerald

2010-10-01

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5-dimethylphenylcarbamate)-coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid-liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors. Copyright © 2010 John Wiley & Sons, Ltd.

Analysis of sesquiterpene lactones, lignans, and flavonoids in wormwood (Artemisia absinthium L.) using high-performance liquid chromatography (HPLC)-mass spectrometry, reversed phase HPLC, and HPLC-solid phase extraction-nuclear magnetic resonance.

PubMed

Aberham, Anita; Cicek, Serhat Sezai; Schneider, Peter; Stuppner, Hermann

2010-10-27

Today, the medicinal use of wormwood (Artemisia absinthium) is enjoying a resurgence of popularity. This study presents a specific and validated high-performance liquid chromatography (HPLC)-diode array detection method for the simultaneous determination and quantification of bioactive compounds in wormwood and commercial preparations thereof. Five sesquiterpene lactones, two lignans, and a polymethoxylated flavonoid were baseline separated on RP-18 material, using a solvent gradient consisting of 0.085% (v/v) o-phosphoric acid and acetonitrile. The flow rate was 1.0 mL/min, and chromatograms were recorded at 205 nm. The stability of absinthin was tested exposing samples to light, moisture, and different temperatures. Methanolic and aqueous solutions of absinthin were found to be stable for up to 6 months. This was also the case when the solid compound was kept in the refrigerator at -35 °C. In contrast, the colorless needles, when stored at room temperature, turned yellow. Three degradation compounds (anabsin, anabsinthin, and the new dimer 3'-hydroxyanabsinthin) were identified by HPLC-mass spectrometry and HPLC-solid-phase extraction-nuclear magnetic resonance and quantified by the established HPLC method.

Quantitative structure-retention relationships applied to development of liquid chromatography gradient-elution method for the separation of sartans.

PubMed

Golubović, Jelena; Protić, Ana; Otašević, Biljana; Zečević, Mira

2016-04-01

QSRR are mathematically derived relationships between the chromatographic parameters determined for a representative series of analytes in given separation systems and the molecular descriptors accounting for the structural differences among the investigated analytes. Artificial neural network is a technique of data analysis, which sets out to emulate the human brain's way of working. The aim of the present work was to optimize separation of six angiotensin receptor antagonists, so-called sartans: losartan, valsartan, irbesartan, telmisartan, candesartan cilexetil and eprosartan in a gradient-elution HPLC method. For this purpose, ANN as a mathematical tool was used for establishing a QSRR model based on molecular descriptors of sartans and varied instrumental conditions. The optimized model can be further used for prediction of an external congener of sartans and analysis of the influence of the analyte structure, represented through molecular descriptors, on retention behaviour. Molecular descriptors included in modelling were electrostatic, geometrical and quantum-chemical descriptors: connolly solvent excluded volume non-1,4 van der Waals energy, octanol/water distribution coefficient, polarizability, number of proton-donor sites and number of proton-acceptor sites. Varied instrumental conditions were gradient time, buffer pH and buffer molarity. High prediction ability of the optimized network enabled complete separation of the analytes within the run time of 15.5 min under following conditions: gradient time of 12.5 min, buffer pH of 3.95 and buffer molarity of 25 mM. Applied methodology showed the potential to predict retention behaviour of an external analyte with the properties within the training space. Connolly solvent excluded volume, polarizability and number of proton-acceptor sites appeared to be most influential paramateres on retention behaviour of the sartans. Copyright © 2015 Elsevier B.V. All rights reserved.

Retinoid quantification by HPLC/MS(n)

NASA Technical Reports Server (NTRS)

McCaffery, Peter; Evans, James; Koul, Omanand; Volpert, Amy; Reid, Kevin; Ullman, M. David

2002-01-01

Retinoic acid (RA) mediates most of the biological effects of vitamin A that are essential for vertebrate survival. It acts through binding to receptors that belong to the nuclear receptor transcription factor superfamily (Mangelsdorf et al. 1994). It is also a highly potent vertebrate teratogen. To determine the function and effects of endogenous and exogenous RA, it is important to have a highly specific, sensitive, accurate, and precise analytical procedure. Current analyses of RA and other retinoids are labor intensive, of poor sensitivity, have limited specificity, or require compatibility with RA reporter cell lines (Chen et al. 1995. BIOCHEM: Pharmacol. 50: 1257-1264; Creech Kraft et al. 1994. BIOCHEM: J. 301: 111-119; Lanvers et al. 1996. J. Chromatogr. B Biomed. Appl. 685: 233-240; Maden et al. 1998. DEVELOPMENT: 125: 4133-4144; Wagner et al. 1992. DEVELOPMENT: 116: 55-66). This paper describes an HPLC/mass spectrometry/mass spectrometry product ion scan (HPLC/MS(n)) procedure for the analysis of retinoids that employs atmospheric pressure chemical ionization MS. The retinoids are separated by normal-phase column chromatography with a linear hexane-isopropanol-dioxane gradient. Each retinoid is detected by a unique series of MS(n) functions set at optimal collision-induced dissociation energy (30% to 32%) for all MS(n) steps. The scan events are divided into three segments, based on HPLC elution order, to maximize the mass spectrometer duty cycle. The all-trans, 9-cis, and 13-cis RA isomers are separated, if desired, by an isocratic hexane-dioxane-isopropanol mobile phase. This paper describes an HPLC/MS(n) procedure possessing high sensitivity and specificity for retinoids.

Direct HPLC separation of beta-aminoester enantiomers on totally synthetic chiral stationary phases.

PubMed

Gasparrini, F; D'Acquarica, I; Villani, C; Cimarelli, C; Palmieri, G

1997-01-01

The direct separation of beta-aminoester enantiomers by HPLC on synthetic chiral stationary phases based on a pi-acidic derivative of trans 1,2-diaminocyclohexane as selector is described. The application of different columns containing the stationary phase with opposite configurations and in the racemic form to the determination of enantiomeric excess in chemically impure samples is demonstrated.

Environmentally evaluated HPLC-ELSD method to monitor enzymatic synthesis of a non-ionic surfactant.

PubMed

Gaber, Yasser; Akerman, Cecilia Orellana; Hatti-Kaul, Rajni

2014-01-01

N-Lauroyl-N-methylglucamide is a biodegradable surfactant derived from renewable resources. In an earlier study, we presented an enzymatic solvent-free method for synthesis of this compound. In the present report, the HPLC method developed to follow the reaction between lauric acid/methyl laurate and N-methyl glucamine (MEG) and its environmental assessment are described. Use of ultraviolet (UV) absorption or refractive index (RI) detectors did not allow the detection of N-methyl glucamine (MEG). With Evaporative light scattering detector ELSD, it was possible to apply a gradient elution, and detect MEG with a limit of detection, LOD = 0.12 μg. A good separation of the peaks: MEG, lauric acid, product (amide) and by-product (amide-ester) was achieved with the gradient program with a run time of 40 min. The setting of ELSD detector was optimized using methyl laurate as the analyte. LC-MS/MS was used to confirm the amide and amide-ester peaks. We evaluated the greenness of the developed method using the freely available software HPLC-Environmental Assessment Tool (HPLC-EAT) and the method got a scoring of 73 HPLC-EAT units, implying that the analytical procedure was more environmentally benign compared to some other methods reported in literature whose HPLC-EAT values scored up to 182. Use of ELSD detector allowed the detection and quantification of the substrates and the reaction products of enzymatic synthesis of the surfactant, N-lauroyl-N-methylglucamide. The developed HPLC method has acceptable environmental profile based on HPLC-EAT evaluation.

Environmentally evaluated HPLC-ELSD method to monitor enzymatic synthesis of a non-ionic surfactant

PubMed Central

2014-01-01

Background N-Lauroyl-N-methylglucamide is a biodegradable surfactant derived from renewable resources. In an earlier study, we presented an enzymatic solvent-free method for synthesis of this compound. In the present report, the HPLC method developed to follow the reaction between lauric acid/methyl laurate and N-methyl glucamine (MEG) and its environmental assessment are described. Results Use of ultraviolet (UV) absorption or refractive index (RI) detectors did not allow the detection of N-methyl glucamine (MEG). With Evaporative light scattering detector ELSD, it was possible to apply a gradient elution, and detect MEG with a limit of detection, LOD = 0.12 μg. A good separation of the peaks: MEG, lauric acid, product (amide) and by-product (amide-ester) was achieved with the gradient program with a run time of 40 min. The setting of ELSD detector was optimized using methyl laurate as the analyte. LC-MS/MS was used to confirm the amide and amide-ester peaks. We evaluated the greenness of the developed method using the freely available software HPLC-Environmental Assessment Tool (HPLC-EAT) and the method got a scoring of 73 HPLC-EAT units, implying that the analytical procedure was more environmentally benign compared to some other methods reported in literature whose HPLC-EAT values scored up to 182. Conclusion Use of ELSD detector allowed the detection and quantification of the substrates and the reaction products of enzymatic synthesis of the surfactant, N-lauroyl-N-methylglucamide. The developed HPLC method has acceptable environmental profile based on HPLC-EAT evaluation. PMID:24914404

Size exclusion chromatography-gradients, an alternative approach to polymer gradient chromatography: 2. Separation of poly(meth)acrylates using a size exclusion chromatography-solvent/non-solvent gradient.

PubMed

Schollenberger, Martin; Radke, Wolfgang

2011-10-28

A gradient ranging from methanol to tetrahydrofuran (THF) was applied to a series of poly(methyl methacrylate) (PMMA) standards, using the recently developed concept of SEC-gradients. Contrasting to conventional gradients the samples eluted before the solvent, i.e. within the elution range typical for separations by SEC, however, the high molar mass PMMAs were retarded as compared to experiments on the same column using pure THF as the eluent. The molar mass dependence on retention volume showed a complex behaviour with a nearly molar mass independent elution for high molar masses. This molar mass dependence was explained in terms of solubility and size exclusion effects. The solubility based SEC-gradient was proven to be useful to separate PMMA and poly(n-butyl crylate) (PnBuA) from a poly(t-butyl crylate) (PtBuA) sample. These samples could be separated neither by SEC in THF, due to their very similar hydrodynamic volumes, nor by an SEC-gradient at adsorbing conditions, due to a too low selectivity. The example shows that SEC-gradients can be applied not only in adsorption/desorption mode, but also in precipitation/dissolution mode without risking blocking capillaries or breakthrough peaks. Thus, the new approach is a valuable alternative to conventional gradient chromatography. Copyright © 2011 Elsevier B.V. All rights reserved.

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

PubMed Central

Mortimer, Monika; Petersen, Elijah J.; Buchholz, Bruce A.; Holden, Patricia A.

2016-01-01

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. The optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation. PMID:27917301

Structural elucidation of potential impurities in Azilsartan bulk drug by HPLC.

PubMed

Zhou, Wentao; Zhou, Yuxia; Sun, Lili; Zou, Qiaogen; Wei, Ping; Ouyang, Pingkai

2014-01-01

During the synthesis of Azilsartan (AZS), it was speculated that 15 potential impurities would arise. This study investigated the possible mechanism for the formation of 14 of them, and their structures were characterized and confirmed by IR, NMR, and MS techniques. In addition, an efficient chromatographic method was developed to separate and quantify these impurities, using an Inertsil ODS-3 column (250 x 4.6 mm, 5 pm) in gradient mode with a mixture of acetonitrile and the potassium dihydrogen orthophosphate buffer (10 mM, pH adjusted to 3.0 with phosphoric acid). The HPLC method was validated for specificity, precision, accuracy, and sensitivity. LOQ of impurities were in the range of 1.04-2.20 ng. Correlation coefficient values of linearity were >0.9996 for AZS and its impurities. The mean recoveries of all impurities in AZS were between 93.0 and 109.7%. Thus, the validated HPLC method is suitable for the separation and quantification of all potential impurities in AZS.

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mortimer, Monika; Petersen, Elijah; Buchholz, Bruce

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here in this paper, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstratemore » separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. Lastly, the optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.« less

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

DOE PAGES

Mortimer, Monika; Petersen, Elijah; Buchholz, Bruce; ...

2016-10-12

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here in this paper, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstratemore » separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. Lastly, the optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.« less

Analysis of munitions constituents in IMX formulations by HPLC and HPLC-MS.

PubMed

Russell, A L; Seiter, J M; Coleman, J G; Winstead, B; Bednar, A J

2014-10-01

The use of Insensitive Munitions eXplosives (IMX) is increasing as the Army seeks to replace certain conventional munitions constituents, such as 2,4,6-trinitrotolene (TNT), for improved safety. The IMX formulations are more stable and therefore less prone to accidental detonation while designed to match the performance of legacy materials. Two formulations, IMX 101 and 104 are being investigated as a replacement for TNT in artillery rounds and composition B Army mortars, respectively. The chemical formulations of IMX-101 and 104 are comprised of four constituents;2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), 1-nitroguanidine (NQ), and Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) which are mixed in various ratios to achieve the desired performance. The current work details the analysis of the IMX constituents by single column HPLC-UV-ESI-MS. Detection limits determined are in agreement with similar HPLC analysis of compounds, ranging from 7 to 9μg/L. Gradient mobile phases are used to allow separation of the 4 target compounds in more complex mixture of other concomitant compounds. Mass spectra are used to confirm analyte identity with chromatographic retention time. Published by Elsevier B.V.

HPLC analysis of functionalized poly(amidoamine) dendrimers and the interaction between a folate-dendrimer conjugate and folate binding protein.

PubMed

Shi, Xiangyang; Bi, Xiangdong; Ganser, T Rose; Hong, Seungpyo; Myc, Lukasz A; Desai, Ankur; Holl, Mark M Banaszak; Baker, James R

2006-07-01

Poly(amidoamine) (PAMAM) dendrimers of different generations with carboxyl, acetyl, and hydroxyl terminal groups and a folic acid (FA)-dendrimer conjugate were separated and analyzed using reverse-phase high performance liquid chromatography (HPLC). Analysis of both the individual PAMAM derivatives and the separation of mixed generations can be achieved using a linear gradient 0-50% acetonitrile (ACN) (balance water) within 40 min. We also show that PAMAMs with defined acetylation and carboxylation degrees can be analyzed using HPLC. Furthermore, a generation 5 dendrimer-FA conjugate (G5.75Ac-FA4; Ac denotes acetyl) was analyzed and its specific binding with a bovine folic acid binding protein (FBP) was monitored. The HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicate the formation of three complexes after the binding of G5.75Ac-FA4 with FBP. Dendrimers with FA moieties show much higher specific binding capability with FBP than those without FA moieties. Findings from this study indicate that HPLC is an effective technique not only for characterization and separation of functionalized PAMAM dendrimers and conjugates but also for investigation of the interaction between dendrimers and biomolecules.

Gradient Self-Doped CuBi2O4 with Highly Improved Charge Separation Efficiency.

PubMed

Wang, Fuxian; Septina, Wilman; Chemseddine, Abdelkrim; Abdi, Fatwa F; Friedrich, Dennis; Bogdanoff, Peter; van de Krol, Roel; Tilley, S David; Berglund, Sean P

2017-10-25

A new strategy of using forward gradient self-doping to improve the charge separation efficiency in metal oxide photoelectrodes is proposed. Gradient self-doped CuBi 2 O 4 photocathodes are prepared with forward and reverse gradients in copper vacancies using a two-step, diffusion-assisted spray pyrolysis process. Decreasing the Cu/Bi ratio of the CuBi 2 O 4 photocathodes introduces Cu vacancies that increase the carrier (hole) concentration and lowers the Fermi level, as evidenced by a shift in the flat band toward more positive potentials. Thus, a gradient in Cu vacancies leads to an internal electric field within CuBi 2 O 4 , which can facilitate charge separation. Compared to homogeneous CuBi 2 O 4 photocathodes, CuBi 2 O 4 photocathodes with a forward gradient show highly improved charge separation efficiency and enhanced photoelectrochemical performance for reduction reactions, while CuBi 2 O 4 photocathodes with a reverse gradient show significantly reduced charge separation efficiency and photoelectrochemical performance. The CuBi 2 O 4 photocathodes with a forward gradient produce record AM 1.5 photocurrent densities for CuBi 2 O 4 up to -2.5 mA/cm 2 at 0.6 V vs RHE with H 2 O 2 as an electron scavenger, and they show a charge separation efficiency of 34% for 550 nm light. The gradient self-doping accomplishes this without the introduction of external dopants, and therefore the tetragonal crystal structure and carrier mobility of CuBi 2 O 4 are maintained. Lastly, forward gradient self-doped CuBi 2 O 4 photocathodes are protected with a CdS/TiO 2 heterojunction and coated with Pt as an electrocatalyst. These photocathodes demonstrate photocurrent densities on the order of -1.0 mA/cm 2 at 0.0 V vs RHE and evolve hydrogen with a faradaic efficiency of ∼91%.

Quantitative and pattern recognition analyses of magnoflorine, spinosin, 6'''-feruloyl spinosin and jujuboside A by HPLC in Zizyphi Semen.

PubMed

Kim, Won Il; Zhao, Bing Tian; Zhang, Hai Yan; Lee, Je Hyun; Son, Jong Keun; Woo, Mi Hee

2014-01-01

Two rapid and simple HPLC methods with UV detector to determine three main compounds (magnoflorine, spinosin and 6'''-feruloyl spinosin) and evaporative light scattering detector (ELSD) to determine jujuboside A were developed for the chemical analyses of Zizyphi Semen. Magnoflorine, spinosin, and 6'''-feruloyl spinosin were separated with an YMC J'sphere ODS-H80 column (250 mm × 4.6 mm, 4 μm) by the gradient elution followed by the isocratic elution using methanol with 0.1 % formic acid and water with 0.1 % formic acid as the mobile phase. The flow rate was 1.0 mL/min. Jujuboside A was separated by HPLC-ELSD with YoungJinBioChrom Aegispak C18-L column (250 mm × 4.6 mm, 5 μm) column in a gradient elution using methanol with 0.1 % formic acid (A) and water with 0.1 % formic acid as the mobile phase. These two methods were fully validated with respect to linearity, precision, accuracy, stability, and robustness. These HPLC methods were applied successfully to quantify four compounds in a Zizyphi Semen extract. The HPLC analytical methods were validated for pattern recognition analysis by repeated analysis of 91 seed samples corresponding to 48 Zizyphus jujuba var. spinosa (J01-J48) and 43 Zizyphus mauritiana (M01-M43). The results indicate that these methods are suitable for a quality evaluation of Zizyphi Semen.

Constant pressure mode extended simple gradient liquid chromatography system for micro and nanocolumns.

PubMed

Šesták, Jozef; Kahle, Vladislav

2014-07-11

Performing gradient liquid chromatography at constant pressure instead of constant flow rate has serious potential for shortening the analysis time and increasing the productivity of HPLC instruments that use gradient methods. However, in the constant pressure mode the decreasing column permeability during a long period of time negatively affects the repeatability of retention time. Thus a volume-based approach, in which the detector signal is plotted as a function of retention volume, must be taken into consideration. Traditional HPLC equipment, however, requires quite complex hardware and software modifications in order to work at constant pressure and in the volume-based mode. In this short communication, a low cost and easily feasible pressure-controlled extension of the previously described simple gradient liquid chromatography platform is proposed. A test mixture of four nitro esters was separated by 10-60% (v/v) acetone/water gradient and a high repeatability of retention volumes at 20MPa (RSD less than 0.45%) was realized. Separations were also performed at different values of pressure (20, 25, and 31MPa), and only small variations of the retention volumes (up to 0.8%) were observed. In this particular case, the gain in the analysis speed of 7% compared to the constant flow mode was realized at a constant pressure. Copyright © 2014 Elsevier B.V. All rights reserved.

Aqueous Reversed-Phase HPLC/FT-IR Using Diffuse Reflectance Detections

NASA Astrophysics Data System (ADS)

Kalasinsky, Victor F.; Pai, T. H.; Kenton, R. C.; Kalasinsky, Kathryn S.

1989-12-01

Solvent-elimination HPLC/FT-IR has become a viable combination of two important techniques, and we have been developing a system which is adaptable to both normal and reversed-phase liquid chromatography. The interface involves the deposition of HPLC eluites onto a KCI-laden train with subsequent analysis via diffuse reflectance spectroscopy, and with minor modifications, the system can be used with microbore and analytical columns. With aqueous solvents, the water is converted to methanol and acetone in a post-column reaction with 2,2-dimethoxypropane before the eluites are deposited. A number of different samples have been used to demonstrate the interface and its flexibility. Steroids, analgesics, and other pharmaceutical preparations have been separated with reverse-phase solvents and identified by their infrared spectra. For some of the compounds studied, different infrared spectra of a given compound have been found to exhibit intensity variations, which arise from different crystalline states. The differences can be concentration dependent and may be useful in obtaining semi-quantitative information from the infrared spectra. Applications involving both gradient elution and isocratic separations have been successful. The former provides the same advantages for HPLC/FT-IR as one finds in conventional HPLC. More recent work has been applied to the use of buffers such as those frequently used in bioanalytical separations. In trying to simplify the post-column reaction with water, we have immobilized dehydration reagents onto silica particles and packed these materials into a column which is inserted in-line after the analytical column. Of the reagents utilized to date, 3,3-dimethoxypropyltrimethoxysilane has been found to perform most efficiently. It has advantages over the simpler reagents because it can be regenerated in the reaction column. Results and the efficiency of the dehydration process and its relation to the type of reagent and its coverage will be

Incorporating high-pressure electroosmotic pump and a nano-flow gradient generator into a miniaturized liquid chromatographic system for peptide analysis.

PubMed

Chen, Apeng; Lynch, Kyle B; Wang, Xiaochun; Lu, Joann J; Gu, Congying; Liu, Shaorong

2014-09-24

We integrate a high-pressure electroosmotic pump (EOP), a nanoflow gradient generator, and a capillary column into a miniaturized liquid chromatographic system that can be directly coupled with a mass spectrometer for proteomic analysis. We have recently developed a low-cost high-pressure EOP capable of generating pressure of tens of thousands psi, ideal for uses in miniaturized HPLC. The pump worked smoothly when it was used for isocratic elutions. When it was used for gradient elutions, generating reproducible gradient profiles was challenging; because the pump rate fluctuated when the pump was used to pump high-content organic solvents. This presents an issue for separating proteins/peptides since high-content organic solvents are often utilized. In this work, we solve this problem by incorporating our high-pressure EOP with a nano-flow gradient generator so that the EOP needs only to pump an aqueous solution. With this combination, we develop a capillary-based nano-HPLC system capable of performing nano-flow gradient elution; the pump rate is stable, and the gradient profiles are reproducible and can be conveniently tuned. To demonstrate its utility, we couple it with either a UV absorbance detector or a mass spectrometer for peptide separations. Copyright © 2014. Published by Elsevier B.V.

Fast Gradient Elution Reversed-Phase HPLC with Diode-Array Detection as a High Throughput Screening Method for Drugs of Abuse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peter W. Carr; K.M. Fuller; D.R. Stoll

A new approach has been developed by modifying a conventional gradient elution liquid chromatograph for the high throughput screening of biological samples to detect the presence of regulated intoxicants. The goal of this work was to improve the speed of a gradient elution screening method over current approaches by optimizing the operational parameters of both the column and the instrument without compromising the reproducibility of the retention times, which are the basis for the identification. Most importantly, the novel instrument configuration substantially reduces the time needed to re-equilibrate the column between gradient runs, thereby reducing the total time for eachmore » analysis. The total analysis time for each gradient elution run is only 2.8 minutes, including 0.3 minutes for column reequilibration between analyses. Retention times standard calibration solutes are reproducible to better than 0.002 minutes in consecutive runs. A corrected retention index was adopted to account for day-to-day and column-to-column variations in retention time. The discriminating power and mean list length were calculated for a library of 47 intoxicants and compared with previous work from other laboratories to evaluate fast gradient elution HPLC as a screening tool.« less

Rapid-gradient HPLC method for measuring drug interactions with immobilized artificial membrane: comparison with other lipophilicity measures.

PubMed

Valko, K; Du, C M; Bevan, C D; Reynolds, D P; Abraham, M H

2000-08-01

A fast-gradient high-performance liquid chromatographic (HPLC) method has been suggested to characterize the interactions of drugs with an immobilized artificial membrane (IAM). With a set of standards, the gradient retention times can be converted to Chromatographic Hydrophobicity Index values referring to IAM chromatography (CHI(IAM)) that approximates an acetonitrile concentration with which the equal distribution of compound can be achieved between the mobile phase and IAM. The CHI(IAM) values are more suitable for interlaboratory comparison and for high throughput screening of new molecular entities than the log k(IAM) values (isocratic retention factor on IAM). The fast-gradient method has been validated against the isocratic log k(IAM) values using the linear free energy relationship solvation equations based on the data from 48 compounds. The compound set was selected to provide a wide range and the least cross-correlation between the molecular descriptors in the solvation equation: (2) where SP is a solute property (e.g., logarithm of partition coefficients, reversed-phase (RP)-HPLC retention parameters, such as log k, log k(w), etc.) and the explanatory variables are solute descriptors as follows: R(2) is an excess molar refraction that can be obtained from the measured refractive index of a compound, pi(2)(H) is the solute dipolarity/polarizability, summation operatoralpha(2)(H) and summation operatorbeta(2)(0) are the solute overall or effective hydrogen-bond acidity and basicity, respectively, and V(x) is the McGowan characteristic volume (in cm(3)/100 mol) that can be calculated for any solute simply from molecular structure using a table of atomic constants. It was found that the relative constants of the solvation equation were very similar for the CHI(IAM) and for the log k(IAM). The IAM lipophilicity scale was quite similar to the octanol/water lipophilicity scale for neutral compounds. The effect of charge on the interaction with IAM was studied

Towards multimodal HPLC separations on humic acid-bonded aminopropyl silica: RPLC and HILIC behavior.

PubMed

Gezici, Orhan; Kara, Hüseyin

2011-09-15

The stationary phase characteristics of the material obtained through immobilization of humic acid (HA) to aminopropyl silica (APS) via amide-bond formation were investigated. The material was characterized in terms of elemental analysis, FTIR, thermogravimetric analyses, pH point of zero charge measurements, potentiometric titrations, and contact angle measurements. Amount of HA bonded to APS was determined from the elemental analysis results, and found as 170 mgHA/gAPS. Stability of the material was studied in aqueous media at different pH values, and amount of HA released at pH=8 did not exceed 2% of the total immobilized HA. Stationary phase characteristics of the well-characterized material were investigated in an HPLC system by using some low-molecular weight polar compounds (i.e. some nucleosides and nucleobases) as test solutes. Effect of some experimental variables such as column conditioning, composition of mobile phase, and temperature on the chromatographic behavior of the studied compounds was studied. Role of ammonium solutions at different pH values on retentive properties of the species was also studied. Retention factors (k') versus volume percentage of organic modifier exhibited a U-curve, which was evaluated as an indication for RPLC/HILIC mixed-mode behavior of the stationary phase. Orthogonality between RPLC and HILIC modes was analyzed through geometric approach, and found as 48.5%. Base-line separation for the studied groups of compounds was achieved under each studied mode, and some differentiations were observed in elution order of the compounds depending on the HPLC mode applied. Chromatograms recorded under RPLC and HILIC modes were compared with those recorded on APS under similar conditions, and thus the influence/importance of HA immobilization process was evaluated in detail. In light of the obtained results, immobilized HA is represented as a useful stationary phase for HPLC separations. Copyright © 2011 Elsevier B.V. All rights

Working principle and application of magnetic separation for biomedical diagnostic at high- and low-field gradients.

PubMed

Leong, Sim Siong; Yeap, Swee Pin; Lim, JitKang

2016-12-06

Magnetic separation is a versatile technique used in sample preparation for diagnostic purpose. For such application, an external magnetic field is applied to drive the separation of target entity (e.g. bacteria, viruses, parasites and cancer cells) from a complex raw sample in order to ease the subsequent task(s) for disease diagnosis. This separation process not only can be achieved via the utilization of high magnetic field gradient, but also, in most cases, low magnetic field gradient with magnitude less than 100 T m -1 is equally feasible. It is the aim of this review paper to summarize the usage of both high gradient magnetic separation and low gradient magnetic separation (LGMS) techniques in this area of research. It is noteworthy that effectiveness of the magnetic separation process not only determines the outcome of a diagnosis but also directly influences its accuracy as well as sensing time involved. Therefore, understanding the factors that simultaneously influence the efficiency of both magnetic separation process and target detection is necessary. Moreover, for LGMS, there are several important considerations that should be taken into account in order to ensure its successful implementation. Hence, this review paper aims to provide an overview to relate all this crucial information by linking the magnetic separation theory to biomedical diagnostic applications.

High-performance liquid-chromatographic separation of subcomponents of antimycin-A

USGS Publications Warehouse

Abidi, S.L.

1988-01-01

Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, Ala, Alb, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins Al, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifiers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpretated based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.

Gradient HPLC of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and pharmaceutical samples using C-18 and RP-amide columns.

PubMed

Kumar, Ashwini; Kumar Malik, Ashok; Kumar Tewary, Dhananjay; Singh, Baldev

2008-02-01

A simple and highly sensitive high pressure liquid chromatographic (HPLC-UV) method has been developed for the determination of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, in mobile phase citrate buffer (0.001 M) of pH 4.5 prepared in water (X), methanol (Y), and ACN (Z) using gradient at a flow rate of 1.0 mL/min by direct UV absorbance detection at lambda = 280 nm. Separation of analytes was studied on the C-18 and RP-amide columns and best results were observed on the RP-amide column with LODs (3.3 x S/m) 0.89, 0.55, 0.67, and 1.41 ng/mL for ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, respectively, and better RSD than the C-18 column. The recovery of Fluoroquinolones (FQs) in urine, ground water, hospital wastewater, and chicken muscle using this method is more than 90%. The method was successfully applied to the analysis of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid in urine, ground water, pharmaceutical dosage forms, hospital wastewater, and chicken muscle.

HPLC-Chip/MS Technology in Proteomic Profiling

NASA Astrophysics Data System (ADS)

Vollmer, Martin; van de Goor, Tom

HPLC-chip/MS is a novel nanoflow analytical technology conducted on a microfabricated chip that allows for highly efficient HPLC separation and superior sensitive MS detection of complex proteomic mixtures. This is possible through on-chip preconcentration and separation with fluidic connection made automatically in a leak-tight fashion. Minimum precolumn and postcolumn peak dispersion and uncompromised ease of use result in compounds eluting in bands of only a few nanoliters. The chip is fabricated out of bio-inert polyimide-containing channels and integrated chip structures, such as an electrospray emitter, columns, and frits manufactured by laser ablation technology. Meanwhile, a variety of HPLC-chips differing in design and stationary phase are commercially available, which provide a comprehensive solution for applications in proteomics, glycomics, biomarker, and pharmaceutical discovery. The HPLC-chip can also be easily integrated into a multidimensional separation workflow where different orthogonal separation techniques are combined to solve a highly complex separation problems. In this chapter, we describe in detail the methodological chip usage and functionality and its application in the elucidation of the protein profile of human nucleoli.

Size-separation of silver nanoparticles using sucrose gradient centrifugation

DOE PAGES

Suresh, Anil K.; Pelletier, Dale A.; Moon, Ji Won; ...

2015-08-28

Size and shape distributions of nanoparticles can drastically contribute to the overall properties of nanoparticles, thereby influencing their interaction with different chemotherapeutic molecules, biological organisms and or materials and cell types. Therefore, to exploit the proper use of nanoparticles for various biomedical and biosensor applications, it is important to obtain well-separated monodispersed nanoparticles. However, gaining precise control over the morphological characteristics of nanoparticles during their synthesis is often a challenging task. Consequently, post-synthesis separation of nanoparticles is necessary. In the present study, we demonstrate the successful one-pot post-synthesis separation of anisotropic silver nanoparticles to near modispersities using sucrose density gradientmore » sedimentation. The separation of the nanoparticles was evidenced based on optical confirmation, and spectrophotometric and transmission electron microscopy measurements. Our results clearly demonstrate the facile separation of anisotropic silver nanoparticles using sucrose density gradient sedimentation and can enable the use of nanoparticles for various biomedical applications.« less

Size-separation of silver nanoparticles using sucrose gradient centrifugation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suresh, Anil K.; Pelletier, Dale A.; Moon, Ji Won

Size and shape distributions of nanoparticles can drastically contribute to the overall properties of nanoparticles, thereby influencing their interaction with different chemotherapeutic molecules, biological organisms and or materials and cell types. Therefore, to exploit the proper use of nanoparticles for various biomedical and biosensor applications, it is important to obtain well-separated monodispersed nanoparticles. However, gaining precise control over the morphological characteristics of nanoparticles during their synthesis is often a challenging task. Consequently, post-synthesis separation of nanoparticles is necessary. In the present study, we demonstrate the successful one-pot post-synthesis separation of anisotropic silver nanoparticles to near modispersities using sucrose density gradientmore » sedimentation. The separation of the nanoparticles was evidenced based on optical confirmation, and spectrophotometric and transmission electron microscopy measurements. Our results clearly demonstrate the facile separation of anisotropic silver nanoparticles using sucrose density gradient sedimentation and can enable the use of nanoparticles for various biomedical applications.« less

Working principle and application of magnetic separation for biomedical diagnostic at high- and low-field gradients

PubMed Central

Yeap, Swee Pin; Lim, JitKang

2016-01-01

Magnetic separation is a versatile technique used in sample preparation for diagnostic purpose. For such application, an external magnetic field is applied to drive the separation of target entity (e.g. bacteria, viruses, parasites and cancer cells) from a complex raw sample in order to ease the subsequent task(s) for disease diagnosis. This separation process not only can be achieved via the utilization of high magnetic field gradient, but also, in most cases, low magnetic field gradient with magnitude less than 100 T m−1 is equally feasible. It is the aim of this review paper to summarize the usage of both high gradient magnetic separation and low gradient magnetic separation (LGMS) techniques in this area of research. It is noteworthy that effectiveness of the magnetic separation process not only determines the outcome of a diagnosis but also directly influences its accuracy as well as sensing time involved. Therefore, understanding the factors that simultaneously influence the efficiency of both magnetic separation process and target detection is necessary. Moreover, for LGMS, there are several important considerations that should be taken into account in order to ensure its successful implementation. Hence, this review paper aims to provide an overview to relate all this crucial information by linking the magnetic separation theory to biomedical diagnostic applications. PMID:27920891

SEPARATION AND PURIFICATION OF TWO MINOR COMPOUNDS FROM RADIX ISATIDIS BY INTEGRATIVE MPLC AND HSCCC WITH PREPARATIVE HPLC.

PubMed

Liang, Zhenjie; Li, Bin; Liang, Yong; Su, Yaping; Ito, Yoichiro

2015-01-01

Radix isatidis has been widely used as a Chinese traditional medicine for its anti-virus and anticancer activities where the minor components may contribute to these beneficial pharmaceutical effects. In order to enrich the target minor compounds effectively and rapidly, extraction, medium-pressure liquid chromatography (MPLC), high-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (pre-HPLC) were integratively used for separation and purification of two target minor compounds indole-3-acetonitrile-6-O-β-D-glucopyranoside (target 1) and clemastanin B (target 2) in the present study. Radix isatidis was dried, pulverized and extracted with 50% methanol at room temperature, then concentrated and subjected to pretreatment with D-101 macroporous resin chromatography and extraction by MPLC. The first target compound was separated by MPLC at the purity raised to 70-80%, but without the second minor compounds which were irreversibly adsorbed by C18 solid support. Therefore, the second target compound in the crude extract was directly separated by HSCCC at purity of 80-90%. Finally these refined samples were further separated by pre-HPLC to obtain a high purity at 98-99%. The chemical structure identification of each target compound was carried out by IR, ESI-MS and 1 H NMR.

Use of shift gradient in the second dimension to improve the separation space in comprehensive two-dimensional liquid chromatography.

PubMed

Li, Duxin; Schmitz, Oliver J

2013-08-01

Comprehensive two-dimensional liquid chromatography (LC × LC) has received much attention because it offers much higher peak capacities than separation in a single dimension. The advantageous peak capacity makes it attractive for the separation of complex samples. Various gradient methods have been used in LC × LC systems. The use of continuous shift gradient is advantageous because it combines the peak compression effect of full gradient mode and the tailed gradient program in parallel gradient mode. Here, a comparison of LC × LC analysis of Chinese herbal medicine with full gradient mode and shift gradient mode in the second dimension was performed. A correlation between the first and second dimensions was found in full gradient mode, and this was significantly reduced with shift gradient mode. The orthogonality increased by 43.7%. The effective peak distribution area increased significantly, which produced better separation.

[HPLC specific chromatogram of Dendrobium officinale].

PubMed

Yan, Mei-Qiu; Chen, Su-Hong; Lv, Gui-Yuan; Zhou, Gui-Fen; Liu, Xia

2013-02-01

To establish the method of specific chromatogram analysis of ether extract of Dendrobium officinale for identification of D. officinale. Chromatographic separation was carried out at 30 degrees C on an Ultimate C18 column (4.6 mm x 250 mm, 5 microm) eluted with methanol and water containing 0.2% phosphoric acid in a gradient elution at a flow rate of 1.0 mL x min(-1). The detection wavelength was set at 280 nm. The similarity evaluation system for chromatographic fingerprint of NPC (National Pharmacopoeia Committee) was adopted to specific chromatogram construction. The HPLC specific chromatogram of D. officinale was constructed with 6 common specific chromatographic peaks including naringenin as a reference peak. The method shows good precision and repeatability of relative retention time. It can be used to identify D. officinale.

[Determination of four alkaloids in Corydalis decumbens by HPLC].

PubMed

Shen, Yan; Han, Chao; Xia, Biqi; Zhou, Yongfang; Liu, Cuiping; Liu, Aili

2011-08-01

To establish a quantitative HPLC method for determination of protopine, palmatine hydrochloride, bicuculline and tetrahydropalmatine, in Corydalis decumbens. The separation was performed on a ZORBAX Eclipse XDB-C18 column (4.6 mm x 150 mm, 5 microm) at a flow rate of 1.0 mL x min(-1) using mixtures of two solvents [A(20 mmol x L(-1) ammonium acetate)-B(acetonitrile)]: with a gradient elution. The column oven temperature was 30 degrees C and the detection wavelength was set at 280 nm. The 4 alkaloids were well separated by this HPLC method. Linearifies of protopine, palmatine hydrochloride, bicuculline and tetrahydropalmatine were good in the ranges of 1.44-46.0 (r = 0.999 4), 1.2640.2 (r = 0.999 8), 1.37-44.0 (r = 0.999 9), and 1.3643.6 mg x L(-1) (r = 0.999 9), respectively. The average recoveries were 98.2% with RSD 2.7% for protopine, 101.9% with RSD 2.5% for palmatine hydrochloride, 102.8% with RSD 3.5% for tetrahydropalmatine, and 98.8% with RSD 3.1% for tetrahydropalmatine. This method is proved to be convenient, reliable and accurate., and it can be used for quality control of C. decumbens.

Optoelectrofluidic field separation based on light-intensity gradients

PubMed Central

Lee, Sanghyun; Park, Hyun Jin; Yoon, Jin Sung; Kang, Kwan Hyoung

2010-01-01

Optoelectrofluidic field separation (OEFS) of particles under light -intensity gradient (LIG) is reported, where the LIG illumination on the photoconductive layer converts the short-ranged dielectrophoresis (DEP) force to the long-ranged one. The long-ranged DEP force can compete with the hydrodynamic force by alternating current electro-osmosis (ACEO) over the entire illumination area for realizing effective field separation of particles. In the OEFS system, the codirectional illumination and observation induce the levitation effect, compensating the attenuation of the DEP force under LIG illumination by slightly floating particles from the surface. Results of the field separation and concentration of diverse particle pairs (0.82–16 μm) are well demonstrated, and conditions determining the critical radius and effective particle manipulation are discussed. The OEFS with codirectional LIG strategy could be a promising particle manipulation method in many applications where a rapid manipulation of biological cells and particles over the entire working area are of interest. PMID:20697461

Optoelectrofluidic field separation based on light-intensity gradients.

PubMed

Lee, Sanghyun; Park, Hyun Jin; Yoon, Jin Sung; Kang, Kwan Hyoung

2010-07-14

Optoelectrofluidic field separation (OEFS) of particles under light -intensity gradient (LIG) is reported, where the LIG illumination on the photoconductive layer converts the short-ranged dielectrophoresis (DEP) force to the long-ranged one. The long-ranged DEP force can compete with the hydrodynamic force by alternating current electro-osmosis (ACEO) over the entire illumination area for realizing effective field separation of particles. In the OEFS system, the codirectional illumination and observation induce the levitation effect, compensating the attenuation of the DEP force under LIG illumination by slightly floating particles from the surface. Results of the field separation and concentration of diverse particle pairs (0.82-16 mum) are well demonstrated, and conditions determining the critical radius and effective particle manipulation are discussed. The OEFS with codirectional LIG strategy could be a promising particle manipulation method in many applications where a rapid manipulation of biological cells and particles over the entire working area are of interest.

Extraction and identification of flavonoids from parsley extracts by HPLC analysis

NASA Astrophysics Data System (ADS)

Stan, M.; Soran, M. L.; Varodi, C.; Lung, I.

2012-02-01

Flavonoids are phenolic compounds isolated from a wide variety of plants, and are valuable for their multiple properties, including antioxidant and antimicrobial activities. In the present work, parsley (Petroselinum crispum L.) extracts were obtained by three different extraction techniques: maceration, ultrasonic-assisted and microwave-assisted solvent extractions. The extractions were performed with ethanol-water mixtures in various ratios. From these extracts, flavonoids like the flavones apigenin and luteolin, and the flavonols quercetin and kaempferol were identified using an HPLC Shimadzu apparatus equipped with PDA and MS detectors. The separation method involved a gradient step. The mobile phase consisted of two solvents: acetonitrile and distilled water with 0.1% formic acid. The separation was performed on a RP-C18 column.

Characterization of Jamaican agro-industrial wastes. Part II, fatty acid profiling using HPLC: precolumn derivatization with phenacyl bromide.

PubMed

Bailey-Shaw, Y A; Golden, K D; Pearson, A G M; Porter, R B R

2012-09-01

This paper describes the determination of fatty acid composition of coffee, citrus and rum distillery wastes using reversed-phase high-performance liquid chromatography (RP-HPLC). Lipid extracts of the waste samples are derivatized with phenacyl bromide and their phenacyl esters are separated on a C8 reversed-phase column by using continuous gradient elution with water and acetonitrile. The presence of saturated and unsaturated fatty acids in quantifiable amounts in the examined wastes, as well as the high percentage recoveries, are clear indications that these wastes have potential value as inexpensive sources of lipids. The HPLC procedures described here could be adopted for further analysis of materials of this nature.

'Click Chemistry' in the preparation of porous polymer-basedparticulate stationary phases for mu-HPLC separation of peptides andproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slater, Michael; Snauko, Marian; Svec, Frantisek

With the use of the copper(I)-catalyzed (3 + 2) azide-alkynecycloaddition, an element of "click chemistry," stationary phasescarrying long alkyl chains or soybean trypsin inhibitor have beenprepared for use in HPLC separations in the reversed-phase and affinitymodes, respectively. The ligands were attached via a triazole ring tosize monodisperse porous beads containing either alkyne or azide pendantfunctionalities. Alkyne-containing beads prepared by directcopolymerization of propargyl acrylate with ethylene dimethacrylate wereallowed to react with azidooctadecane to give a reversed-phase sorbent.Azide-functionalized beads were prepared by chemical modification ofglycidyl methacrylate particles. Subsequent reaction with a terminalaliphatic alkyne produced a reversed-phase sorbent similar to thatobtained from themore » alkyne beads. Soybean trypsin inhibitor wasfunctionalized with N-(4-pentynoyloxy) succinimide to carry alkyne groupsand then allowed to react with the azide-containing beads to produce anaffinity sorbent for trypsin. The performance of these stationary phaseswas demonstrated with the HPLC separations of a variety of peptides andproteins.« less

Separation of foreground and background from light field using gradient information.

PubMed

Lee, Jae Young; Park, Rae-Hong

2017-02-01

Studies of computer vision or machine vision applications using a light field camera have been increasing in recent years. However, the abilities that the light field camera has are not fully used in these applications. In this paper, we propose a method for direct separation of foreground and background that uses the gradient information and can be used in various applications such as pre-processing. From an optical phenomenon whereby the bundles of rays from the background are flipped, we derive that the disparity sign of the background in the captured three-dimensional scene has the opposite disparity sign of the foreground. Using the majority-weighted voting algorithm based on the gradient information with the Lambertian assumption and the gradient constraint, the foreground and background can be separated at each pixel. In regard to pre-processing, the proposed method can be used for various applications such as occlusion and saliency detection, disparity estimation, and so on. Experimental results with the EPFL light field dataset and Stanford Lytro light field dataset show that the proposed method achieves better performance in terms of the occlusion detection, and thus can be effectively used in pre-processing for saliency detection and disparity estimation.

Quantification of appetite suppressing steroid glycosides from Hoodia gordonii in dried plant material, purified extracts and food products using HPLC-UV and HPLC-MS methods.

PubMed

Janssen, Hans-Gerd; Swindells, Chris; Gunning, Philip; Wang, Weijun; Grün, Christian; Mahabir, Krishna; Maharaj, Vinesh J; Apps, Peter J

2008-06-09

High-performance liquid chromatography (HPLC)-UV and HPLC-Mass Spectrometry (MS) methods were developed for the quantitative analysis of the family of Hoodia gordonii steroid glycosides with appetite suppressing properties in dried plant material, in purified and enriched extracts and in various prototype food-products fortified with H. gordonii extracts. For solid materials, e.g. dried plants or for non-fatty foods, extraction of the steroid glycosides is performed using methanol. For products where the steroid glycosides are present in an oil matrix, direct injection of the oil after dilution in tetrahydrofuran is applied. The HPLC separation is performed on an octyl-modified reversed-phase column in the gradient mode with UV detection at lambda = 220 nm. Quantification is performed against an external calibration line prepared using either one of the pure steroid glycosides or geranyl-tiglate. Short- and long-term repeatabilities of the methods are better than 3 and 6%, respectively. Recoveries are better than 85%, even in the analysis of the least abundant steroid glycosides in a complex yoghurt drink. Linearity is better than 3-4 orders of magnitude and the detection limits are below approximately 2 microg g(-1) for the individual steroid glycosides in dried plant material and food products. HPLC-MS is used to confirm that the steroid glycosides contain the characteristic steroid core, the carbohydrate chain and the tigloyl group.

Simultaneous analysis of eight bioactive compounds in Danning tablet by HPLC-ESI-MS and HPLC-UV.

PubMed

Liu, Runhui; Zhang, Jiye; Liang, Mingjin; Zhang, Weidong; Yan, Shikai; Lin, Min

2007-02-19

A high performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ESI-MS) and ultraviolet detector (UV) has been developed for the simultaneous analysis of eight bioactive compounds in Danning tablet (including hyperin, hesperidin, resveratrol, nobiletin, curcumine, emodin, chrysophanol, and physcion), a widely used prescription of traditional Chinese medicine (TCM). The chromatographic separation was performed on a ZORBAX Extend C(18) analytical column by gradient elution with acetonitrile and formate buffer (containing 0.05% formic acid, adjusted with triethylamine to pH 5.0) at a flow rate of 0.8 ml/min. The eight compounds in Danning tablet were identified and their MS(n) fractions were elucidated by using HPLC-ESI-MS, and the contents of these compounds were determined by using HPLC-UV method. The standard calibration curves were linear between 5.0 and 100 microg/ml for hyperin, 10-200 microg/ml for hesperidin, 1.0-150 microg/ml for resveratrol, 2.0-120 microg/ml for nobiletin, 2.0-225 microg/ml for curcumine, 20-300 microg/ml for emodin, 2.0-200 microg/ml for chrysophanol, and 20-250 microg/ml for physcion with regression coefficient r(2)>0.9995. The intra-day and inter-day precisions of this method were evaluated with the R.S.D. values less than 0.7% and 1.3%, respectively. The recoveries of the eight investigated compounds were ranged from 99.3% to 100.2% with R.S.D. values less than 1.5%. This method was successfully used to determine the 8 target compounds in 10 batches of Danning tablet.

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families.

PubMed

Bremner, P D; Blacklock, C J; Paganga, G; Mullen, W; Rice-Evans, C A; Crozier, A

2000-06-01

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.

Disaggregation and separation dynamics of magnetic particles in a microfluidic flow under an alternating gradient magnetic field

NASA Astrophysics Data System (ADS)

Cao, Quanliang; Li, Zhenhao; Wang, Zhen; Qi, Fan; Han, Xiaotao

2018-05-01

How to prevent particle aggregation in the magnetic separation process is of great importance for high-purity separation, while it is a challenging issue in practice. In this work, we report a novel method to solve this problem for improving the selectivity of size-based separation by use of a gradient alternating magnetic field. The specially designed magnetic field is capable of dynamically adjusting the magnetic field direction without changing the direction of magnetic gradient force acting on the particles. Using direct numerical simulations, we show that particles within a certain center-to-center distance are inseparable under a gradient static magnetic field since they are easy aggregated and then start moving together. By contrast, it has been demonstrated that alternating repulsive and attractive interaction forces between particles can be generated to avoid the formation of aggregations when the alternating gradient magnetic field with a given alternating frequency is applied, enabling these particles to be continuously separated based on size-dependent properties. The proposed magnetic separation method and simulation results have the significance for fundamental understanding of particle dynamic behavior and improving the separation efficiency.

[Study on HPLC fingerprint of Oldenlandia diffusa].

PubMed

Chen, Yan; Yao, Zhi-Hong; Dai, Yi; Cheng, Hong; Wen, Li-Rong; Zhou, Guang-Xiong; Yao, Xin-Sheng

2012-06-01

To establish the HPLC fingerprint chromatogram of Oldenlandia diffusa coupled with chemometrics means for the quality control of multi-batches of medicinal material. The separation was developed on C18 column(4.6 mm x 250 mm, 5 microm) by gradient elution with acetonitrile-water(both containing 0.1 per thousand (V/V) ocetic acid) as mobile phase at a flow rate of 0.8 mL/min, the detection wavelength at 238 nm and column temperature at 30 degrees C. The HPLC fingerprint chromatogram of Oldenlandia diffusa was set up and the main characteristic peaks were identified by comparing with chemical reference substance. The quality of 22 batches of medicinal material was evaluated by similarity assay as well as principal component analysis (PCA) and cluster analysis. The established HPLC fingerprint chromatogram of Oldenlandia diffusa was specific, precise, reproducible and stable. 11 peaks were chemically identified. The similarity of 17 batches of Oldenlandia diffusa was obviously higher than 5 batches of adulterants. PCA showed that 17 batches of Oldenlandia diffusa were in a domain and 5 batches of adulterants were far apart from the domain. The cluster analysis of the 22 batches of medicinal material showed that 17 batches of Oldenlandia diffusa were in a cluster while 5 batches of adulterants were excluded. Further cluster analysis was carried out for the quality consistency of 17 batches of Oldenlandia diffusa and accordingly they were devided into 4 clusters. With the combination of chemometrics means, the HPLC fingerprint chromatogram provides a method for evaluation of authenticity and quality control of Oldenlandia diffusa, which is favorable to improve overall quality control of Oldenlandia diffusa.

Direct injection analysis of fatty and resin acids in papermaking process waters by HPLC/MS.

PubMed

Valto, Piia; Knuutinen, Juha; Alén, Raimo

2011-04-01

A novel HPLC-atmospheric pressure chemical ionization/MS (HPLC-APCI/MS) method was developed for the rapid analysis of selected fatty and resin acids typically present in papermaking process waters. A mixture of palmitic, stearic, oleic, linolenic, and dehydroabietic acids was separated by a commercial HPLC column (a modified stationary C(18) phase) using gradient elution with methanol/0.15% formic acid (pH 2.5) as a mobile phase. The internal standard (myristic acid) method was used to calculate the correlation coefficients and in the quantitation of the results. In the thorough quality parameters measurement, a mixture of these model acids in aqueous media as well as in six different paper machine process waters was quantitatively determined. The measured quality parameters, such as selectivity, linearity, precision, and accuracy, clearly indicated that, compared with traditional gas chromatographic techniques, the simple method developed provided a faster chromatographic analysis with almost real-time monitoring of these acids. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chiral separation with gradient elution isotachophoresis for future in situ extraterrestrial analysis.

PubMed

Danger, Grégoire; Ross, David

2008-10-01

The first results of chiral separations with the gradient elution isotachophoresis method are presented. As previously described, citrate is used in the run buffer as the leading ion and borate in the sample buffer as the terminating ion. Modulation of parameters such as electrolyte pH, pressure scan rate, chiral selector concentration, combinations of CD or the percentage of ampholytes provides an easy optimization of the separations. To perform fluorescent detection 5-carboxyfluorescein succinimidyl ester and two fluorogenic-labeling agents, fluorescamine (Fluram) and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde, are used to label amino acids. With the 5-carboxyfluorescein amino acids, chiral separations are easily obtained using a neutral CD ((2-hydroxypropyl)-beta-CD) at a low concentration (2 mmol/L). With Fluram amino acids, the situation is more complicated due to the formation of diastereoisomers and due to weak interactions with the different CDs used. The use of the 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde-labeling agent solves the problems observed with the Fluram agent while retaining the fluorogenic properties. These first results demonstrate the simplicity and the feasibility of gradient elution isotachophoresis for chiral separations.

Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC.

PubMed

Cheel, José; Urajová, Petra; Hájek, Jan; Hrouzek, Pavel; Kuzma, Marek; Bouju, Elodie; Faure, Karine; Kopecký, Jiří

2017-02-01

Puwainaphycins are a recently described group of β-amino fatty acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium (Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n-hexane-ethyl acetate-ethanol-water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min -1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate-ethanol-water (5:1:5, v/v/v) system, afforded compounds 1 and 2. The CCC separation of fraction II, with use of the n-hexane-ethyl acetate-ethanol-water (1:5:1:5, v/v/v/v) system, afforded compounds 3-7. In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min -1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1-7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC-electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1-7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1, 2, and 5) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass. Graphical Abstract Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Compounds 1 (12-hydroxy-4-methyl

High gradient magnetic field microstructures for magnetophoretic cell separation.

PubMed

Abdel Fattah, Abdel Rahman; Ghosh, Suvojit; Puri, Ishwar K

2016-08-01

Microfluidics has advanced magnetic blood fractionation by making integrated miniature devices possible. A ferromagnetic microstructure array that is integrated with a microfluidic channel rearranges an applied magnetic field to create a high gradient magnetic field (HGMF). By leveraging the differential magnetic susceptibilities of cell types contained in a host medium, such as paramagnetic red blood cells (RBCs) and diamagnetic white blood cells (WBCs), the resulting HGMF can be used to continuously separate them without attaching additional labels, such as magnetic beads, to them. We describe the effect of these ferromagnetic microstructure geometries have on the blood separation efficacy by numerically simulating the influence of microstructure height and pitch on the HGMF characteristics and resulting RBC separation. Visualizations of RBC trajectories provide insight into how arrays can be optimized to best separate these cells from a host fluid. Periodic microstructures are shown to moderate the applied field due to magnetic interference between the adjacent teeth of an array. Since continuous microstructures do not similarly weaken the resultant HGMF, they facilitate significantly higher RBC separation. Nevertheless, periodic arrays are more appropriate for relatively deep microchannels since, unlike continuous microstructures, their separation effectiveness is independent of depth. The results are relevant to the design of microfluidic devices that leverage HGMFs to fractionate blood by separating RBCs and WBCs. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification and anti-oxidant capacity determination of phenolics and their glycosides in elderflower by on-line HPLC-CUPRAC method.

PubMed

Çelik, S Esin; Özyürek, Mustafa; Güçlü, Kubilay; Çapanoğlu, Esra; Apak, Reşat

2014-01-01

Development and application of an on-line cupric reducing anti-oxidant capacity (CUPRAC) assay coupled with HPLC for separation and on-line determination of phenolic anti-oxidants in elderflower (Sambucus nigra L.) extracts for their anti-oxidant capacity are significant for evaluating health-beneficial effects. Moreover, this work aimed to assay certain flavonoid glycosides of elderflower that could not be identified/quantified by other similar on-line HPLC methods (i.e. 2,2-diphenyl-1-picrylhdrazyl and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid). To identify anti-oxidant constituents in elderflower by HPLC and to evaluate their individual anti-oxidant capacities by on-line HPLC-CUPRAC assay with a post-column derivatisation system. The separation and UV detection of polyphenols were performed on a C18 -column using gradient elution with two different mobile phase solutions, that is acetonitrile and 1% glacial acetic acid, with detection at 340 nm. The HPLC-separated anti-oxidant polyphenols in column effluent react with copper(II)-neocuproine in a reaction-coil to reduce the latter to copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The detection limits of tested compounds at 450 nm after post-column derivatisation were compared with those of at 340 nm UV-detection without derivatisation. LOD values (µg/mL) of quercetin and its glycosides at 450 nm were lower than those of UV detection at 340 nm. This method was applied successfully to elderflower extract. The flavonol glycosides of quercetin and kaempferol bound to several sugar components (glucose, rhamnose, galactose and rutinose) were identified in the sample. The on-line HPLC-CUPRAC method was advantageous over on-line ABTS and DPPH methods for measuring the flavonoid glycosides of elderflower. Copyright © 2014 John Wiley & Sons, Ltd.

Analyses of procyanidins in foods using Diol phase HPLC

USDA-ARS?s Scientific Manuscript database

Separation of procyanidins using silica-based HPLC suffered from poor resolution for higher oligomers and low sensitivity due to the fluorescence quenching effects of methylene chloride in the mobile phase. Optimization of a published Diol-phase HPLC method resulted in near baseline separation for p...

Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectral identification of anthocyanins and flavonols in Shiraz grape skin.

PubMed

Downey, Mark O; Rochfort, Simone

2008-08-01

A limitation of large-scale viticultural trials is the time and cost of comprehensive compositional analysis of the fruit by high-performance liquid chromatography (HPLC). In addition, separate methods have generally been required to identify and quantify different classes of metabolites. To address these shortcomings a reversed-phase HPLC method was developed to simultaneously separate the anthocyanins and flavonols present in grape skins. The method employs a methanol and water gradient acidified with 10% formic acid with a run-time of 48 min including re-equilibration. Identity of anthocyanins and flavonols in Shiraz (Vitis vinifera L.) skin was confirmed by mass spectral analysis.

Accessible silanol sites - beneficial for the RP-HPLC separation of constitutional and diastereomeric azaspirovesamicol isomers.

PubMed

Wenzel, Barbara; Fischer, Steffen; Brust, Peter; Steinbach, Jörg

2010-12-10

Different RP-HPLC columns (phenyl, conventional ODS, cross-linked C(18) and special end-capped C(8) and C(18) phases) were used to investigate the separation of four basic ionizable isomers. Using ACN/20mM NH(4)OAc aq., a separation was observed exclusively on RP columns with higher silanol activity at unusual high ACN concentration, indicating cation-exchange as main retention mechanism. Using MeOH/20mM NH(4)OAc aq., another separation at low MeOH concentrations was observed on both, RP columns with higher as well as RP columns with lower silanol activity, which is mainly based on hydrophobic interactions. The isomers were also separated on a bare silica column at higher MeOH content using NH(4)OAc. Since cation-exchange governs this retention, the elution order was different compared to the RP phases. A strong retention on the silica column was observed in ACN, which could be attributed to partition processes as additional retention mechanism. Copyright © 2010 Elsevier B.V. All rights reserved.

Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate.

PubMed

Xie, Rui; Tu, Maobing; Wu, Yonnie; Adhikari, Sushil

2011-04-01

5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase. The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30 °C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45 °C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did. Published by Elsevier Ltd.

Microwave-assisted extraction with water for fast extraction and simultaneous RP-HPLC determination of phenolic acids in radix Salviae Miltiorrhizae.

PubMed

Fang, Xinsheng; Wang, Jianhua; Zhou, Hongying; Jiang, Xingkai; Zhu, Lixiang; Gao, Xin

2009-07-01

An optimized microwave-assisted extraction method using water (MAE-W) as the extractant and an efficient HPLC analysis method were first developed for the fast extraction and simultaneous determination of D(+)-(3,4-dihydroxyphenyl) lactic acid (Dla), salvianolic acid B (SaB), and lithospermic acid (La) in radix Salviae Miltiorrhizae. The key parameters of MAE-W were optimized. It was found that the degradation of SaB was inhibited when using the optimized MAE-W and the stable content of Dla, La, and SaB in danshen was obtained. Furthermore, compared to the conventional extraction methods, the proposed MAE-W is a more rapid method with higher yield and lower solvent consumption with a reproducibility (RSD <6%). In addition, using water as extractant is safe and helpful for environment protection, which could be referred to as green extraction. The separation and quantitative determination of the three compounds was carried out by a developed reverse-phase high-performance liquid chromatographic (RP-HPLC) method with UV detection. Highly efficient separation was obtained using gradient solvent system. The optimized HPLC analysis method was validated to have specificity, linearity, precision, and accuracy. The results indicated that MAE-W followed by HPLC-UV determination is an appropriate alternative to previously proposed method for quality control of radix Salviae Miltiorrhizae.

Simultaneous Estimation of Withaferin A and Z-Guggulsterone in Marketed Formulation by RP-HPLC.

PubMed

Agrawal, Poonam; Vegda, Rashmi; Laddha, Kirti

2015-07-01

A simple, rapid, precise and accurate high-performance liquid chromatography (HPLC) method was developed for simultaneous estimation of withaferin A and Z-guggulsterone in a polyherbal formulation containing Withania somnifera and Commiphora wightii. The chromatographic separation was achieved on a Purosphere RP-18 column (particle size 5 µm) with a mobile phase consisting of Solvent A (acetonitrile) and Solvent B (water) with the following gradients: 0-7 min, 50% A in B; 7-9 min, 50-80% A in B; 9-20 min, 80% A in B at a flow rate of 1 mL/min and detection at 235 nm. The marker compounds were well separated on the chromatogram within 20 min. The results obtained indicate accuracy and reliability of the developed simultaneous HPLC method for the quantification of withaferin A and Z-guggulsterone. The proposed method was found to be reproducible, specific, precise and accurate for simultaneous estimation of these marker compounds in a combined dosage form. The HPLC method was appropriate and the two markers are well resolved, enabling efficient quantitative analysis of withaferin A and Z-guggulsterone. The method can be successively used for quantitative analysis of these two marker constituents in combination of marketed polyherbal formulation. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides

PubMed Central

Park, Sung-Soo; Maudsley, Stuart

2010-01-01

Global profiling of phosphoproteomes has proven a great challenge due to the relatively low stoichiometry of protein phosphorylation and poor ionization efficiency in mass spectrometers. Effective, physiologically-relevant, phosphoproteome research relies on the efficient phosphopeptide enrichment from complex samples. Immobilized metal affinity chromatography and titanium dioxide chromatography (TOC) can greatly assist selective phosphopeptide enrichment. However, the complexity of resultant enriched samples is often still high, suggesting that further separation of enriched phosphopeptides is required. We have developed a pH-gradient elution technique for enhanced phosphopeptide identification in conjunction with TOC. Using this process, we have demonstrated its superiority to the traditional ‘one-pot’ strategies for differential protein identification. Our technique generated a highly specific separation of phosphopeptides by an applied pH-gradient between 9.2 and 11.3. The most efficient elution range for high-resolution phosphopeptide separation was between pH 9.2 and 9.4. High-resolution separation of multiply-phosphorylated peptides was primarily achieved using elution ranges > pH 9.4. Investigation of phosphopeptide sequences identified in each pH fraction indicated that phosphopeptides with phosphorylated residues proximal to acidic residues, including glutamic acid, aspartic acid, and other phosphorylated residues, were preferentially eluted at higher pH values. PMID:20946866

Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC.

PubMed

Tarasova, Irina A; Goloborodko, Anton A; Perlova, Tatyana Y; Pridatchenko, Marina L; Gorshkov, Alexander V; Evreinov, Victor V; Ivanov, Alexander R; Gorshkov, Mikhail V

2015-07-07

The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC.

Separation and quantitation of colour pigments of chili powder (Capsicum frutescens) by high-performance liquid chromatography-diode array detection.

PubMed

Cserháti, T; Forgács, E; Morais, M H; Mota, T; Ramos, A

2000-10-27

The performance of reversed-phase thin-layer (RP-TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) was compared for the separation and determination of the colour pigments of chili (Capsicum frutescens) powder using a wide variety of eluent systems. No separation of pigments was achieved in RP-TLC, however, it was established that tetrahydrofuran shows an unusually high solvent strength. RP-HPLC using water-methanol-acetonitrile gradient elution separated the chili pigments in many fractions. Diode array detection (DAD) indicated that yellow pigments are eluted earlier than the red ones and chili powder contains more yellow pigments than common paprika powders. It was established that the very different absorption spectra of pigments make the use of DAD necessary.

Separation of aromatic carboxylic acids using quaternary ammonium salts on reversed-phase HPLC. 1. Separation behavior of aromatic carboxylic acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawamura, K.; Okuwaki, A.; Verheyen, T.

In order to develop separation processes and analytical methods for aromatic carboxylic acids for the coal oxidation products, the separation behavior of aromatic carboxylic acids on a reversed-phase HPLC using eluent containing quaternary ammonium salt has been investigated. The retention mechanism of aromatic carboxylic acids was discussed on the basis of both ion-pair partition model and ion-exchange model. The retention behavior of aromatic carboxylic acids possessing one (or two) carboxylic acid group(s) followed the ion-pair partition model, where linear free energy relationship was observed between the capacity factor and the extraction equilibrium constants of benzoic acid and naphthalene carboxylic acid.more » Besides, the retention behavior followed ion-exchange model with increasing the number of carboxylic acids, where the capacity factor of benzene polycarboxylic acids is proportional to the association constants between aromatic acids and quaternary ammonium ions calculated on the basis of an electrostatic interaction model.« less

Micropower Mixed-signal VLSI Independent Component Analysis for Gradient Flow Acoustic Source Separation.

PubMed

Stanaćević, Milutin; Li, Shuo; Cauwenberghs, Gert

2016-07-01

A parallel micro-power mixed-signal VLSI implementation of independent component analysis (ICA) with reconfigurable outer-product learning rules is presented. With the gradient sensing of the acoustic field over a miniature microphone array as a pre-processing method, the proposed ICA implementation can separate and localize up to 3 sources in mild reverberant environment. The ICA processor is implemented in 0.5 µm CMOS technology and occupies 3 mm × 3 mm area. At 16 kHz sampling rate, ASIC consumes 195 µW power from a 3 V supply. The outer-product implementation of natural gradient and Herault-Jutten ICA update rules demonstrates comparable performance to benchmark FastICA algorithm in ideal conditions and more robust performance in noisy and reverberant environment. Experiments demonstrate perceptually clear separation and precise localization over wide range of separation angles of two speech sources presented through speakers positioned at 1.5 m from the array on a conference room table. The presented ASIC leads to a extreme small form factor and low power consumption microsystem for source separation and localization required in applications like intelligent hearing aids and wireless distributed acoustic sensor arrays.

Determination of CMPO using HPLC -UV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gracy Elias; Gary S. Groenewold; Bruce J. Mincher

Octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide (CMPO) is an extractant proposed for selective separation of radionuclide metals from used nuclear fuel solutions using solvent extraction. Radiolysis reactions can degrade CMPO and reduce separation performance and hence methods for measuring concentration of CMPO and identifying degradation products are needed. A novel high performance liquid chromatography (HPLC) method employing ultraviolet detection (UV) was developed to detect and quantitate CMPO in dodecane. Some radiolysis products in gamma and alpha irradiated CMPO solutions were identified using HPLC/electrospray ionization-mass spectrometry (ESI-MS). Validation data indicated that the HPLC-UV method for CMPO determination provided good linearity, sensitivity, procedure accuracy and systemmore » precision. CMPO-nitric acid complexes were also identified, that account for the apparent loss of CMPO in acidic environment, independent of irradiation.« less

A chiral HPLC method for the simultaneous separation of configurational isomers of the predominant cis/trans forms of astaxanthin.

PubMed

Abu-Lafi, S; Turujman, S A

1997-01-01

We report an HPLC method that allows the simultaneous separation of configurational isomers of the predominant cis/trans forms of astaxanthin. The configurational isomers of the all-trans-, and most of the configurational isomers of the 9-cis-, 13-cis- and 15-cis-astaxanthin were separated on a Sumichiral OA-2000 column, which is manufactured and packed in Japan with a Pirkle covalent D-phenylglycine chiral stationary phase (CSP). The large separation of the cis isomers from the all-trans isomers that we report here ensure the suitability of this method for the routine determination of the ratio of the configurational isomers of all-trans-astaxanthin.

Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS.

PubMed

Rodríguez-Medina, Inmaculada C; Beltrán-Debón, Raúl; Molina, Vicente Micol; Alonso-Villaverde, Carlos; Joven, Jorge; Menéndez, Javier A; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

2009-10-01

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD-HPLC-ESI-TOF-MS and IT-MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF-MS in order to obtain molecular formula and exact mass. Posterior analyses with IT-MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.

HPLC-DAD-MS identification of bioactive secondary metabolites from Ferula communis roots.

PubMed

Arnoldi, Lolita; Ballero, Mauro; Fuzzati, Nicola; Maxia, Andrea; Mercalli, Enrico; Pagni, Luca

2004-06-01

A simple HPLC method was developed to distinguish between 'poisonous' and 'non-poisonous' chemotypes of Ferula communis. The method was performed on a C8 reverse phase analytical column using a binary eluent (aqueous TFA 0.01%-TFA 0.01% in acetonitrile) under gradient condition. The two chemotypes showed different fingerprints. The identification of five coumarins and eleven daucane derivatives by HPLC-diode array detection (HPLC-DAD) and HPLC-MS is described. A coumarin, not yet described, was detected. Copyright 2004 Elsevier B.V.

Preparation, characterization, and performance evaluation of UiO-66 analogues as stationary phase in HPLC for the separation of substituted benzenes and polycyclic aromatic hydrocarbons.

PubMed

Zhao, Weiwei; Zhang, Chaoyan; Yan, Zengguang; Zhou, Youya; Li, Jianrong; Xie, Yabo; Bai, Liping; Jiang, Lin; Li, Fasheng

2017-01-01

UiO-66 analogues are good candidates as stationary phase in HPLC because of their chemical/thermal stability, large surface area, and two cage structures. Here, two UiO-66 analogues, UiO-66-NH2 and UiO-67, were synthesized and used as stationary phase in HPLC to evaluate their performance in the separation of substituted benzenes (SBs) and polycyclic aromatic hydrocarbons (PAHs). The results showed that SBs could be well separated on UiO-66-NH2 column but not on UiO-67 column. Nonetheless, PAHs could be well separated on UiO-67 column. The separation mechanisms of SBs and PAHs on UiO-66 analogues may be involved in the pore size and functional group in the frameworks of UiO-66 analogues. Introduction of the-NH2 into UiO-66 significantly reduced its adsorption capacity for SB congeners, which resulted in less separation of SBs on UiO-66-NH2. As for the separation of PAHs on UiO-67 column, the π-π stacking effect was supposed to play a vital role.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

PubMed

Llambias, E B; Luo, J

1996-01-01

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

Effects of surface roughness on an adverse-pressure-gradient separating turbulent boundary layer

NASA Astrophysics Data System (ADS)

Wu, Wen; Piomelli, Ugo; Turbulence Simulation; Modelling Laboratory Team

2017-11-01

Separating turbulent boundary layers over smooth and rough flat plates are investigated by large-eddy simulations. A suction-blowing velocity distribution is imposed at the top boundary to produce an adverse-to-favourable pressure gradient and a closed separation bubble. Sandgrain roughness in the fully-rough regime is modelled by an immersed boundary method. In the rough-wall case, streamline detachment occurs earlier and the separation region is substantially larger due to the momentum deficit caused by the roughness. The adverse pressure gradient decreases the form drag and causes a thin reversed-flow region below the roughness crest, so that Cf = 0 does not coincide with the detachment of the flow from the surface. The wake regions behind roughness elements affect the intermittency of the near-wall flow, so that upstream of the detachment point the flow can be reversed half of the time, but its average is positive. The separated shear layer exhibits higher turbulent kinetic energy (TKE); the growth of the TKE there begins earlier relative to the separation point, and the peak TKE occurs close to the separation point. The momentum deficit caused by the roughness, again, plays a critical role in these changes. The authors acknowledge the support from Hydro-Québec and the NSERC Collaborative Research & Development program (CRDPJ 418786-11). The simulations were performed at CAC Queen't site. UP also thanks the support of Canada Research Chair Program.

Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine.

PubMed

Douša, Michal; Srbek, Jan; Rádl, Stanislav; Cerný, Josef; Klecán, Ondřej; Havlíček, Jaroslav; Tkadlecová, Marcela; Pekárek, Tomáš; Gibala, Petr; Nováková, Lucie

2014-06-01

Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC-HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4'-diamino-6,6'-bis[(4-fluorobenzyl)amino]biphenyl-3,3'-diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Tessellated permanent magnet circuits for flow-through, open gradient separations of weakly magnetic materials.

PubMed

Moore, Lee R; Williams, P Stephen; Chalmers, Jeffrey J; Zborowski, Maciej

2017-04-01

Emerging microfluidic-based cell assays favor label-free red blood cell (RBC) depletion. Magnetic separation of RBC is possible because of the paramagnetism of deoxygenated hemoglobin but the process is slow for open-gradient field configurations. In order to increase the throughput, periodic arrangements of the unit magnets were considered, consisting of commercially available Nd-Fe-B permanent magnets and soft steel flux return pieces. The magnet design is uniquely suitable for multiplexing by magnet tessellation, here meaning the tiling of the magnet assembly cross-sectional plane by periodic repetition of the magnet and the flow channel shapes. The periodic pattern of magnet magnetizations allows a reduction of the magnetic material per channel with minimal distortion of the field cylindrical symmetry inside the magnet apertures. A number of such magnet patterns are investigated for separator performance, size and economy with the goal of designing an open-gradient magnetic separator capable of reducing the RBC number concentration a hundred-fold in 1 mL whole blood per hour.

Determination of blood cyanide by HPLC-MS.

PubMed

Tracqui, A; Raul, J S; Géraut, A; Berthelon, L; Ludes, B

2002-04-01

An original high-performance liquid chromatographic-mass spectrometric (HPLC-MS) procedure was developed for the determination of cyanide (CN) in whole blood. After the addition of K13C15N as internal standard, blood was placed in a microdiffusion device, the inner well of which was filled with a mixture of taurine (50mM in water)/naphthalene-2,3-dicarboxaldehyde (NDA, 10mM in methanol)/methanol/ concentrated (approximately 20%) ammonia solution (25:25:45:5, v/v). Concentrated H2SO4 was added to the blood sample, and the microdiffusion chamber was sealed. After 30 min of gentle agitation, 2 microL of the contents of the inner vial were pipetted and directly injected onto a NovaPak C18 HPLC column. Separation was performed by a gradient of acetonitrile in 2mM NH4COOH, pH 3.0 buffer (35-80% in 10 min). Detection was done with a Perkin-Elmer Sciex API-100 mass analyzer with an ionspray interface, operated in the negative ionization mode. MS data were collected as either TIC or SIM at m/z (299 + 191) and (301 + 193) for the derivatives formed with CN and 13C15N, respectively. Inspired by previous works dealing with the complexation of CN by NDA + taurine to form a 1-cyano [f] benzoisoindole derivative analyzed by HPLC-fluorimetry, this method appears simple, rapid, and extremely specific. Limits of detection and quantitation for blood CN are 5 and 15 ng/mL, respectively. The use of 13C15N as internal standard allows the quantitation of CN with elegance and accuracy in comparison with previously reported methods.

HPLC for quality control of polyimides

NASA Technical Reports Server (NTRS)

Young, P. R.; Sykes, G. F.

1979-01-01

High Pressure Liquid Chromatography (HPLC) as a quality control tool for polyimide resins and prepregs are presented. A data base to help establish accept/reject criteria for these materials was developed. This work is intended to supplement, not replace, standard quality control tests normally conducted on incoming resins and prepregs. To help achieve these objectives, the HPLC separation of LARC-160 polyimide precursor resin was characterized. Room temperature resin aging effects were studied. Graphite reinforced composites made from fresh and aged resin were fabricated and tested to determine if changes observed by HPLC were significant.

Separation and characterization of antioxidants from Spirulina platensis microalga combining pressurized liquid extraction, TLC, and HPLC-DAD.

PubMed

Jaime, Laura; Mendiola, José A; Herrero, Miguel; Soler-Rivas, Cristina; Santoyo, Susana; Señorans, F Javier; Cifuentes, Alejandro; Ibáñez, Elena

2005-11-01

A new procedure has been developed to separate and characterize antioxidant compounds from Spirulina platensis microalga based on the combination of pressurized liquid extraction (PLE) and different chromatographic procedures, such as TLC, at preparative scale, and HPLC with a diode array detector (DAD). Different solvents were tested for PLE extraction of antioxidants from S. platensis microalga. An optimized PLE process using ethanol (generally recognized as safe, GRAS) as extraction solvent has been obtained that provides natural extracts with high yields and good antioxidant properties. TLC analysis of this ethanolic extract obtained at 115 degrees C for 15 min was carried out and the silica layer was stained with a DPPH (diphenyl-pycril-hydrazyl) radical solution to determine the antioxidant activity of different chromatographic bands. Next, these colored bands were collected for their subsequent analysis by HPLC-DAD, revealing that the compounds with the most important antioxidant activity present in Spirulina extracts were carotenoids, as well as phenolic compounds and degradation products of chlorophylls.

[Separation and identification of red pigments in natural red yolk of duck's eggs by HPLC-MS-MS].

PubMed

Liu, Liangzhong; Zhang, Min; Peng, Guanghua; Wang, Haibin; Zhang, Shenghua

2004-05-01

The natural red yolk of duck's eggs is produced by the laying duck in the lake areas in southward of China. In the laying duck breeding areas such as Honghu, Jianli, Xiantao, Tianmen and Hanchuan citys in Hubei Province, the culturists are used to feeding fresh pondweeds to the laying ducks. The yolk of duck's eggs is natural red with the chrominance reaching up to and/or above RCF (Roche Yolk Color Fan) 15. The red pigment components of natural red yolk of duck's eggs were separated and identified by thin layer chromatography (TLC), high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS-MS) and high resolution electron impact-mass spectrometry (EI-MS). Four isomers of red pigments were separated by HPLC on a RP-C18 column with methanol-water (99.5:0.5, v/v) as mobile phase. The lambda(max) of the four components were 482, 488, 496, 501 nm, respectively, and all of them were single peak on chromatogram. They had the same molecular mass (Mr = 562), and had the same fragment peaks of MS2 with rhodoxanthin. The molecular formula of red pigments was determined as C40H50O2 by high resolution EI-MS. The results indicate that the red pigment is rhodoxanthin, and they are all cis-isomers of rhodoxanthin.

Preparation, characterization, and performance evaluation of UiO-66 analogues as stationary phase in HPLC for the separation of substituted benzenes and polycyclic aromatic hydrocarbons

PubMed Central

Yan, Zengguang; Li, Jianrong; Xie, Yabo; Bai, Liping; Jiang, Lin; Li, Fasheng

2017-01-01

UiO-66 analogues are good candidates as stationary phase in HPLC because of their chemical/thermal stability, large surface area, and two cage structures. Here, two UiO-66 analogues, UiO-66-NH2 and UiO-67, were synthesized and used as stationary phase in HPLC to evaluate their performance in the separation of substituted benzenes (SBs) and polycyclic aromatic hydrocarbons (PAHs). The results showed that SBs could be well separated on UiO-66-NH2 column but not on UiO-67 column. Nonetheless, PAHs could be well separated on UiO-67 column. The separation mechanisms of SBs and PAHs on UiO-66 analogues may be involved in the pore size and functional group in the frameworks of UiO-66 analogues. Introduction of the–NH2 into UiO-66 significantly reduced its adsorption capacity for SB congeners, which resulted in less separation of SBs on UiO-66-NH2. As for the separation of PAHs on UiO-67 column, the π-π stacking effect was supposed to play a vital role. PMID:28582453

Determination of hydroxyurea in human plasma by HPLC-UV using derivatization with xanthydrol.

PubMed

Legrand, Tiphaine; Rakotoson, Marie-Georgine; Galactéros, Frédéric; Bartolucci, Pablo; Hulin, Anne

2017-10-01

A simple and rapid high performance liquid chromatography (HPLC) method using ultraviolet (UV) detection was developed to determine hydroxyurea (HU) concentration in plasma sample after derivatization with xanthydrol. Two hundred microliters samples were spiked with methylurea (MeU) as internal standard and proteins were precipitated by adding methanol. Derivatization of HU and MeU was immediately performed by adding 0.02M xanthydrol and 1.5M HCl in order to obtain xanthyl-derivatives of HU and MeU that can be further separated using HPLC and quantified using UV detection at 240nm. Separation was achieved using a C18 column with a mobile phase composed of 20mM ammonium acetate and acetonitrile in gradient elution mode at a flow rate of 1mL/min. The total analysis time did not exceed 18min. The method was found linear from 5 to 400μM and all validation parameters fulfilled the international requirements. Between- and within-run accuracy error ranged from -4.7% to 3.2% and precision was lower than 12.8%. This simple method requires small volume samples and can be easily implemented in most clinical laboratories to develop pharmacokinetics studies of HU and to promote its therapeutic monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS.

PubMed

Woo, H; Kim, J W; Han, K M; Lee, J H; Hwang, I S; Lee, J H; Kim, J; Kweon, S J; Cho, S; Chae, K R; Han, S Y; Kim, J

2013-01-01

In order to test health foods for illegally added diuretics for weight loss, we developed simple, rapid, selective, and sensitive methods using HPLC and LC-MS/MS for the simultaneous analysis of 17 diuretics in dietary supplements. HPLC conditions were set with a Capcell-pak C18, using a mobile phase consisting of gradient conditions, UV detection at 254 nm and validated for linearity (r(2)> 0.999), precision (CV ≤ 3%), recoveries (90.4-102.8%) and reproducibility. Identification and quantification of 17 diuretics were accomplished by ion-spray LC-MS/MS using multiple reaction monitoring (MRM). The chromatographic separation was carried out under the reversed-phase mechanism on an HSS-T3 column. The LC-MS/MS method was validated for linearity (r(2)> 0.99) and precision (CV < 13%). Sixteen dietary supplements were tested with the developed methods. Diuretics were not detected in all samples. Extraction recovery was also investigated and the extraction recoveries in different formulations were from 88% to 110% and from 81% to 116% using HPLC and LC-MS/MS, respectively. There was no significant difference in recoveries in the type of dietary supplements. Based on this result, the developed methods to monitor illegal drug adulterations in dietary supplements using HPLC and LC-MS/MS are simple, fast and reliable. Therefore, it is applicable to routine drug-adulteration screening.

Very high pressure liquid chromatography using core-shell particles: quantitative analysis of fast gradient separations without post-run times.

PubMed

Stankovich, Joseph J; Gritti, Fabrice; Stevenson, Paul G; Beaver, Lois A; Guiochon, Georges

2014-01-17

Five methods for controlling the mobile phase flow rate for gradient elution analyses using very high pressure liquid chromatography (VHPLC) were tested to determine thermal stability of the column during rapid gradient separations. To obtain rapid separations, instruments are operated at high flow rates and high inlet pressure leading to uneven thermal effects across columns and additional time needed to restore thermal equilibrium between successive analyses. The purpose of this study is to investigate means to minimize thermal instability and obtain reliable results by measuring the reproducibility of the results of six replicate gradient separations of a nine component RPLC standard mixture under various experimental conditions with no post-run times. Gradient separations under different conditions were performed: constant flow rates, two sets of constant pressure operation, programmed flow constant pressure operation, and conditions which theoretically should yield a constant net heat loss at the column's wall. The results show that using constant flow rates, programmed flow constant pressures, and constant heat loss at the column's wall all provide reproducible separations. However, performing separations using a high constant pressure with programmed flow reduces the analysis time by 16% compared to constant flow rate methods. For the constant flow rate, programmed flow constant pressure, and constant wall heat experiments no equilibration time (post-run time) was required to obtain highly reproducible data. Copyright © 2013 Elsevier B.V. All rights reserved.

Practical utilization of spICP-MS to study sucrose density gradient centrifugation for the separation of nanoparticles.

PubMed

Johnson, Monique E; Montoro Bustos, Antonio R; Winchester, Michael R

2016-11-01

Single particle inductively coupled plasma mass spectrometry (spICP-MS) is shown to be a practical technique to study the efficacy of rate-zonal sucrose density gradient centrifugation (SDGC) separations of mixtures of gold nanoparticles (AuNPs) in liquid suspension. spICP-MS enabled measurements of AuNP size distributions and particle number concentrations along the gradient, allowing unambiguous evaluations of the effectiveness of the separation. Importantly, these studies were conducted using AuNP concentrations that are directly relevant to environmental studies (sub ng mL -1 ). At such low concentrations, other techniques [e.g., dynamic light scattering (DLS), transmission and scanning electron microscopies (TEM and SEM), UV-vis spectroscopy, atomic force microscopy (AFM)] do not have adequate sensitivity, highlighting the inherent value of spICP-MS for this and similar applications. In terms of the SDGC separations, a mixture containing three populations of AuNPs, having mean diameters of 30, 80, and 150 nm, was fully separated, while separations of two other mixtures (30, 60, 100 nm; and 20, 50, 100 nm) were less successful. Finally, it is shown that the separation capacity of SDGC can be overwhelmed when particle number concentrations are excessive, an especially relevant finding in view of common methodologies taken in nanotechnology research. Graphical Abstract Characterization of the separation of a gold nanoparticle mixture by sucrose density gradient centrifugation by conventional and single particle ICP-MS analysis.

Determination of the design space of the HPLC analysis of water-soluble vitamins.

PubMed

Wagdy, Hebatallah A; Hanafi, Rasha S; El-Nashar, Rasha M; Aboul-Enein, Hassan Y

2013-06-01

Analysis of water-soluble vitamins has been tremendously approached through the last decades. A multitude of HPLC methods have been reported with a variety of advantages/shortcomings, yet, the design space of HPLC analysis of these vitamins was not defined in any of these reports. As per the food and drug administration (FDA), implementing the quality by design approach for the analysis of commercially available mixtures is hypothesized to enhance the pharmaceutical industry via facilitating the process of analytical method development and approval. This work illustrates a multifactorial optimization of three measured plus seven calculated influential HPLC parameters on the analysis of a mixture containing seven common water-soluble vitamins (B1, B2, B6, B12, C, PABA, and PP). These three measured parameters are gradient time, temperature, and ternary eluent composition (B1/B2) and the seven calculated parameters are flow rate, column length, column internal diameter, dwell volume, extracolumn volume, %B (start), and %B (end). The design is based on 12 experiments in which, examining of the multifactorial effects of these 3 + 7 parameters on the critical resolution and selectivity, was carried out by systematical variation of all these parameters simultaneously. The 12 basic runs were based on two different gradient time each at two different temperatures, repeated at three different ternary eluent compositions (methanol or acetonitrile or a mixture of both). Multidimensional robust regions of high critical R(s) were defined and graphically verified. The optimum method was selected based on the best resolution separation in the shortest run time for a synthetic mixture, followed by application on two pharmaceutical preparations available in the market. The predicted retention times of all peaks were found to be in good match with the virtual ones. In conclusion, the presented report offers an accurate determination of the design space for critical resolution in the

HPLC Separation of Sulforaphane Enantiomers in Broccoli and Its Sprouts by Transformation into Diastereoisomers Using Derivatization with (S)-Leucine.

PubMed

Okada, Makiko; Yamamoto, Atsushi; Aizawa, Sen-Ichi; Taga, Atsushi; Terashima, Hiroyuki; Kodama, Shuji

2017-01-11

Racemic sulforaphane, which was derivatized with (S)-leucine (l-leucine), was resolved by reversed phase HPLC with UV detection. The optimum mobile phase conditions were found to be 10 mM citric acid (pH 2.8) containing 22% methanol at 35 °C using detection at 254 nm. Sulforaphane enantiomers in florets and stems of five brands of broccoli and leaves and stems of three brands of broccoli sprouts were analyzed by the proposed HPLC method. Both sulforaphane enantiomers were detected in all of the samples. The S/R ratios of sulforaphane in broccoli samples were 1.5-2.6/97.4-98.5% for florets and 5.0-12.1/87.9-95.0% for stems. The S/R ratios in broccoli sprout samples were higher than those in broccoli samples and were found to be 8.3-19.7/80.3-91.7% for leaves and 37.0-41.8/58.2-63.0% for stems. (S)-Sulforaphane detected in the broccoli and its sprout samples was positively identified by separately using an HPLC with a chiral column (Chiralpak AD-RH) and mass spectrometry.

Tessellated permanent magnet circuits for flow-through, open gradient separations of weakly magnetic materials

PubMed Central

Moore, Lee R.; Williams, P. Stephen; Chalmers, Jeffrey J.; Zborowski, Maciej

2017-01-01

Emerging microfluidic-based cell assays favor label-free red blood cell (RBC) depletion. Magnetic separation of RBC is possible because of the paramagnetism of deoxygenated hemoglobin but the process is slow for open-gradient field configurations. In order to increase the throughput, periodic arrangements of the unit magnets were considered, consisting of commercially available Nd-Fe-B permanent magnets and soft steel flux return pieces. The magnet design is uniquely suitable for multiplexing by magnet tessellation, here meaning the tiling of the magnet assembly cross-sectional plane by periodic repetition of the magnet and the flow channel shapes. The periodic pattern of magnet magnetizations allows a reduction of the magnetic material per channel with minimal distortion of the field cylindrical symmetry inside the magnet apertures. A number of such magnet patterns are investigated for separator performance, size and economy with the goal of designing an open-gradient magnetic separator capable of reducing the RBC number concentration a hundred-fold in 1 mL whole blood per hour. PMID:29104346

Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves

NASA Astrophysics Data System (ADS)

Zeb, Alam; Ullah, Fareed

2017-04-01

Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight carotenoids, four pheophytins and two chlorophylls. Lutein (806.0 µg/g), chlorophyll b' (410.0 µg/g), chlorophyll a (162.4 µg/g), 9'-Z-neoxanthin (142.8 µg/g) and all-E-violaxanthin (82.2 µg/g)) were present in higher amounts. The relatively high amounts of lutein may be one of the key indicator of beneficial antioxidant properties. The phenolic profile revealed a total of thirteen compounds, namely p-hydroxybenzoic acid, chlorogenic acid, caftaric acid, kaempferol glycosides, p-coumaric acid, rosmarinic acid, 5-caffeoylquinic acid, and quercetin glycosides. Kaempferol-3-sophorotrioside (58.7 mg/g), chlorogenic acid (28.5 mg/g), 5-O-caffeoylquinic acid (18.7 mg/g), coumaric acid (11.2 mg/g) and its derivative (33.1 mg/g) were present in high amounts. These results suggest that the reversed phase HPLC profiling of adiantum leaves provides a better understanding in to the actual composition of bioactive compounds, which may be responsible for possible medicinal properties. Adiantum leaves rich in important bioactive phytochemicals can be used as a potential source of nutraceuticals or as a functional food ingredient.

Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves

PubMed Central

Zeb, Alam; Ullah, Fareed

2017-01-01

Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight carotenoids, four pheophytins, and two chlorophylls. Lutein (806.0 μg/g), chlorophyll b′ (410.0 μg/g), chlorophyll a (162.4 μg/g), 9′-Z-neoxanthin (142.8 μg/g) and all-E-violaxanthin (82.2 μg/g) were present in higher amounts. The relatively high amounts of lutein may be one of the key indicator of beneficial antioxidant properties. The phenolic profile revealed a total of 13 compounds, namely 4-hydroxybenzoic acid, chlorogenic acid, caftaric acid, kaempferol glycosides, p-coumaric acid, rosmarinic acid, 5-caffeoylquinic acid, and quercetin glycosides. Kaempferol-3-sophorotrioside (58.7 mg/g), chlorogenic acid (28.5 mg/g), 5-O-caffeoylquinic acid (18.7 mg/g), coumaric acid (11.2 mg/g), and its derivative (33.1 mg/g) were present in high amounts. These results suggest that the reversed phase HPLC profiling of Adiantum leaves provides a better understanding in to the actual composition of bioactive compounds, which may be responsible for the potential medicinal properties. Adiantum leaves rich in important bioactive phytochemicals can be used as a possible source of nutraceuticals or as a functional food ingredient. PMID:28497036

Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves.

PubMed

Zeb, Alam; Ullah, Fareed

2017-01-01

Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight carotenoids, four pheophytins, and two chlorophylls. Lutein (806.0 μg/g), chlorophyll b ' (410.0 μg/g), chlorophyll a (162.4 μg/g), 9'- Z -neoxanthin (142.8 μg/g) and all- E -violaxanthin (82.2 μg/g) were present in higher amounts. The relatively high amounts of lutein may be one of the key indicator of beneficial antioxidant properties. The phenolic profile revealed a total of 13 compounds, namely 4-hydroxybenzoic acid, chlorogenic acid, caftaric acid, kaempferol glycosides, p-coumaric acid, rosmarinic acid, 5-caffeoylquinic acid, and quercetin glycosides. Kaempferol-3-sophorotrioside (58.7 mg/g), chlorogenic acid (28.5 mg/g), 5- O -caffeoylquinic acid (18.7 mg/g), coumaric acid (11.2 mg/g), and its derivative (33.1 mg/g) were present in high amounts. These results suggest that the reversed phase HPLC profiling of Adiantum leaves provides a better understanding in to the actual composition of bioactive compounds, which may be responsible for the potential medicinal properties. Adiantum leaves rich in important bioactive phytochemicals can be used as a possible source of nutraceuticals or as a functional food ingredient.

Long-Gradient Separations Coupled with Selected Reaction Monitoring for Highly Sensitive, Large Scale Targeted Protein Quantification in a Single Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Fillmore, Thomas L.; Gao, Yuqian

2013-10-01

Long-gradient separations coupled to tandem MS were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional LC-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in LOQ for target proteins in human female serum. Based on at least one surrogate peptide per protein, an LOQ ofmore » 10 ng/mL was achieved for the two spiked proteins in non-depleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or sub-ng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and ELISA measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM offers at least 3 times higher multiplexing capacity than conventional LC-SRM due to ~3-fold increase in average peak widths for a 300-min gradient compared to a 45-min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.« less

Comparative studies on performance of CCC and preparative RP-HPLC in separation and purification of steroid saponins from Dioscorea zingiberensis C.H.Wright.

PubMed

Zhang, Xinxin; Liang, Jinru; Zhang, Yongmin; Liu, Jianli; Sun, Wenji; Ito, Yoichiro

2015-03-01

Steroid saponins from Dioscorea zingiberensis C.H.Wright were separated for the first time using two chromatographic methods for comparison: counter-current chromatography (CCC) coupled with evaporative light scattering detector (ELSD) and preparative reversed phase high-performance liquid chromatography (RP-HPLC) with an ultraviolet detector. Ethyl acetate-n-butanol-methanol-water (4:1:2:4, v/v) was chosen as the two-phase solvent system for CCC, while the acetonitrile-water (25:75 for the first step and15:85 for the second step, v/v) was used as the mobile phase in the preparative RP-HPLC. The following five steroid saponins were purified by theses two chromatographic methods, in one-step operation by CCC and by two-step operation in preparative RP-HPLC: 1) 26-O-β-D- glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 26-triol-3-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside ( compound A ), 2) 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 4) 26-triol-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside ( compound B ), 3) 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 26-triol-3-O-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside ( compound C ), 4) 26-O-β-D-glucopyranosyl-(25R)-furost-5, 20(22)-diene-3β, 26-diol-3-O-{α-L-rhamnopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→2)]}-β-D-glucopyranoside ( compound D ) and 5) 26-O-β-D-glucopyranosyl-(25R)-furost-5, 20(22)-diene-3β, 26-diol-3-O-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosy-(1→2)]-β-D-glucopyranoside ( compound E ). The purities of these five steroid saponins separated by both methods were over 95%, and structural identification of these compounds was performed by ESI-MS, and 13 C NMR. Comparison of these two established approaches revealed that CCC required a longer separation time but with less solvent consumption, whereas preparative RP-HPLC gave a shorter

Separation Dynamics of Controlled Internal Flow in an Adverse Pressure Gradient

NASA Astrophysics Data System (ADS)

Peterson, C. J.; Vukasinovic, B.; Glezer, A.

2017-11-01

The effects of fluidic actuation on the dynamic evolution of aggressive internal flow separation is investigated at speeds up to M = 0.4 within a constant-width diffuser branching off of a primary flow duct. It is shown that a spanwise array of fluidic actuators upstream of the separation actively controls the flow constriction (and losses) within the diffuser and consequently the local pressure gradient at its entrance. The effectiveness of the actuation, as may be measured by the increased flow rate that is diverted through the diffuser, scales with its flow rate coefficient. In the presence of actuation (0.7% mass fraction), the mass flow rate in the primary duct increases by 10% while the fraction of the diverted mass flow rate in the diffuser increases by more than 45%. The flow dynamics near separation in the absence and presence of actuation are characterized using high speed particle image velocimetry and analyzed using proper orthogonal and spectral decompositions. In particular, the spectral contents of the incipient boundary layer separation are compared in the absence and presence of actuation with emphasis on the changes in local dynamics near separation as the characteristic cross stream scale of the boundary layer increases with separation delay.

Determination of fructooligosaccharides in burdock using HPLC and microwave-assisted extraction.

PubMed

Li, Jing; Liu, Xiaomei; Zhou, Bin; Zhao, Jing; Li, Shaoping

2013-06-19

The root of burdock ( Arctium lappa L.) is a commonly used vegetable in Asia. Fructooligosaccharides (FOS) are usually considered as its main bioactive components. Thus, quantitative analysis of these components is very important for the quality control of burdock. In this study, an HPLC-ELSD and microwave-assisted extraction method was developed for the simultaneous determination of seven FOS with degrees of polymerization (DP) between 3 and 9, as well as fructose, glucose, and sucrose in burdock from different regions. The separation was performed on a Waters XBridge Amide column (4.6 × 250 mm i.d., 3.5 μm) with gradient elution. All calibration curves for investigated analytes showed good linear regression (r > 0.9990). Their LODs and LOQs were lower than 3.63 and 24.82 μg/mL, respectively. The recoveries ranged from 99.2 to 102.6%. The developed method was successfully applied to determination of ten sugars in burdock from different locations of Asia. The results showed that the contents of FOS in different samples of burdock collected at appropriate times were similar, and the developed HPLC-ELSD with microwave-assisted extraction method is helpful to control the quality of burdock.

Quantitative Magnetic Separation of Particles and Cells using Gradient Magnetic Ratcheting

PubMed Central

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-01-01

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting (MACS), are robust but perform coarse, qualitative separations based on surface antigen expression. We report a quantitative magnetic separation technology using high-force magnetic ratcheting over arrays of magnetically soft micro-pillars with gradient spacing, and use the system to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micro-pillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic-field. Particles with higher IOC separate and equilibrate along the miro-pillar array at larger pitches. We develop a semi-analytical model that predicts behavior for particles and cells. Using the system, LNCaP cells were separated based on the bound quantity of 1μm anti-EpCAM particles as a metric for expression. The ratcheting cytometry system was able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof of concept, EpCAM-labeled cells from patient blood were isolated with 74% purity, demonstrating potential towards a quantitative magnetic separation instrument. PMID:26890496

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

PubMed

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-04-13

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Serum of Blood Separation by Means of Dynamic Electrochromatography in a Centrifugal and Electromagnetic Gradient.

PubMed

Jan Dyszkiewicz, Andrzej

2014-01-01

The paper presents the dynamic association of several gradients of separation with electrophoresis and the laboratory implementation of a prototype device for dynamic electrochromatography in a centrifugal and electromagnetic gradient (DECGOE), based on patent PL171643. The object of the study was to compare the separation capacity and repeatability of the location of edge parameters (xx(1), yy(1), d) of the contours of a proteinogram fraction for conventional electrophoresis (EF), radial SR-DECGOE separation and three variants of radial-transverse SR-SPM, SR-SPE, SRSPRRM distribution as well as the reproducibility of the following prototype parameters: PV(1), PV(2), PV(3)[V] potentials, PV(3)[F] frequency, ω(1) and ω(2) rotational speed, MC mixer chamber buffer [pH] and volume[ml], and B [T] magnetic induction. Human blood serum was used in the study, 50 samples for each separation type. The highest rate of fraction formation was registered for the SR-SPRRM (8,07/s) variant. The percentage values of standard deviations for the edge parameters of proteinograms were the lowest for conventional electrophoresis (EF), slightly higher for radial distribution SR, while among the radial-transverse separations, the highest accuracy was obtained for the SR-SPRRM variant.

[Study on HPLC fingerprint of flavonoids from Houttuynia cordata by comparing with fingerprint reference].

PubMed

Zhang, Ting-Ting; Wu, Yi; Hang, Tai-Jun

2009-05-01

To establish a stable and repeatable HPLC fingerprint standard and evaluate the flavonoids from Houttuynia cordata qualitatively and quantitatively. HPLC separation was performed on a C18 column with methanol-0.1% phosphoric acid mixed solution as mobile phase in gradient elution mode. The fingerprint reference was determined as one of the most typical chromatograms and used to be compared with other samples through Cosine and Relative Euclid Distance methods, thus the chromatographic fingerprints of flavonoids from Houttuynia cordata were evaluated by constitutes and contents, respectively. Fourteen mutual peaks were fixed in the HPLC fingerprint of flavonoids from Houttaynia cordata. It showed good results in validation tests in which the quercitrin's peak was set as the reference peak to calculate relative retention time and area of other peaks in the chromatograms, and the RSD were less than 0.2% and 5.0%, respectively. The linear ranges for quercitrin was 1.07-83.4 microg/mL (r=0.9999) and the average recovery was 100.3%. The method shows good repeatability, ruggedness and reliability. Comparing with the established reference fingerprint, the evaluation system including Cosine and Relative Euclid Distance methods lays dependable foundation for controlling the quality of Houttuynia cordata.

Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity.

PubMed

Valko, Klara; Nunhuck, Shenaz; Bevan, Chris; Abraham, Michael H; Reynolds, Derek P

2003-11-01

A fast gradient HPLC method (cycle time 15 min) has been developed to determine Human Serum Albumin (HSA) binding of discovery compounds using chemically bonded protein stationary phases. The HSA binding values were derived from the gradient retention times that were converted to the logarithm of the equilibrium constants (logK HSA) using data from a calibration set of molecules. The method has been validated using literature plasma protein binding data of 68 known drug molecules. The method is fully automated, and has been used for lead optimization in more than 20 company projects. The HSA binding data obtained for more than 4000 compounds were suitable to set up global and project specific quantitative structure binding relationships that helped compound design in early drug discovery. The obtained HSA binding of known drug molecules were compared to the Immobilized Artificial Membrane binding data (CHI IAM) obtained by our previously described HPLC-based method. The solvation equation approach has been used to characterize the normal binding ability of HSA, and this relationship shows that compound lipophilicity is a significant factor. It was found that the selectivity of the "baseline" lipophilicity governing HSA binding, membrane interaction, and octanol/water partition are very similar. However, the effect of the presence of positive or negative charges have very different effects. It was found that negatively charged compounds bind more strongly to HSA than it would be expected from the lipophilicity of the ionized species at pH 7.4. Several compounds showed stronger HSA binding than can be expected from their lipophilicity alone, and comparison between predicted and experimental binding affinity allows the identification of compounds that have good complementarities with any of the known binding sites. Copyright 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2236-2248, 2003

Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS.

PubMed

Chandra, A; Rana, J; Li, Y

2001-08-01

A method has been established and validated for identification and quantification of individual, as well as total, anthocyanins by HPLC and LC/ES-MS in botanical raw materials used in the herbal supplement industry. The anthocyanins were separated and identified on the basis of their respective M(+) (cation) using LC/ES-MS. Separated anthocyanins were individually calculated against one commercially available anthocyanin external standard (cyanidin-3-glucoside chloride) and expressed as its equivalents. Amounts of each anthocyanin calculated as external standard equivalent were then multiplied by a molecular-weight correction factor to afford their specific quantities. Experimental procedures and use of a molecular-weight correction factors are substantiated and validated using Balaton tart cherry and elderberry as templates. Cyanidin-3-glucoside chloride has been widely used in the botanical industry to calculate total anthocyanins. In our studies on tart cherry and elderberry, its use as external standard followed by use of molecular-weight correction factors should provide relatively accurate results for total anthocyanins, because of the presence of cyanidin as their major anthocyanidin backbone. The method proposed here is simple and has a direct sample preparation procedure without any solid-phase extraction. It enables selection and use of commercially available anthocyanins as external standards for quantification of specific anthocyanins in the sample matrix irrespective of their commercial availability as analytical standards. It can be used as a template and applied for similar quantification in several anthocyanin-containing raw materials for routine quality control procedures, thus providing consistency in analytical testing of botanical raw materials used for manufacturing efficacious and true-to-the-label nutritional supplements.

Chiral separation of isoxanthohumol and 8-prenylnaringenin in beer, hop pellets, and hops by HPLC with chiral columns.

PubMed

Moriya, Hyuga; Tanaka, Sohei; Iida, Yukari; Kitagawa, Satomi; Aizawa, Sen-Ichi; Taga, Atsushi; Terashima, Hiroyuki; Yamamoto, Atsushi; Kodama, Shuji

2018-05-16

Xanthohumol, isoxanthohumol, and 8-prenylnaringenin in beer, hop, and hop pellet samples were analyzed by HPLC using InertSustain phenyl column and the mobile phase containing 40% methanol and 12% 2-propanol. Fractions of isoxanthohumol and 8-prenylnaringenin obtained by the above HPLC were separately collected. Isoxanthohumol and 8-prenylnaringenin were enantioseparated by HPLC using Chiralcel OD-H column with a mobile phase composed of hexane/ethanol (90/10, v/v) and Chiralpak AD-RH column with a mobile phase composed of methanol/2-propanol/water (40/20/40, v/v/v), respectively. Both of isoxanthohumol and 8-prenylnaringenin from beer, hop, and hop pellet samples were found to be a racemic mixture. This can be explained that the two analytes were produced by non-enzymatic process. The effects of boiling conditions on the conversion of xanthohumol into isoxanthohumol were also studied. A higher concentration of ethanol in heating solvent resulted in a decrease in the conversion ratio and the conversion was stopped by addition of ethanol more than 50% (v/v). The isomerization was significantly affected pH (2-10) and the boiling medium at pH 5 was minimum for the conversion. Therefore, it was suggested that xanthohumol was relatively difficult to convert to isoxanthohumol in wort (pH 5-5.5) during boiling. This article is protected by copyright. All rights reserved.

Simultaneous quantification of flavonoids and triterpenoids in licorice using HPLC.

PubMed

Wang, Yuan-Chuen; Yang, Yi-Shan

2007-05-01

Numerous bioactive compounds are present in licorice (Glycyrrhizae Radix), including flavonoids and triterpenoids. In this study, a reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous quantification of three flavonoids (liquiritin, liquiritigenin and isoliquiritigenin) and four triterpenoids (glycyrrhizin, 18alpha-glycyrrhetinic acid, 18beta-glycyrrhetinic acid and 18beta-glycyrrhetinic acid methyl ester) from licorice was developed, and further, to quantify these 7 compounds from 20 different licorice samples. Specifically, the reverse-phase HPLC was performed with a gradient mobile phase composed of 25 mM phosphate buffer (pH 2.5)-acetonitrile featuring gradient elution steps as follows: 0 min, 100:0; 10 min, 80:20; 50 min, 70:30; 73 min, 50:50; 110 min, 50:50; 125 min, 20:80; 140 min, 20:80, and peaks were detected at 254 nm. By using our technique, a rather good specificity was obtained regarding to the separation of these seven compounds. The regression coefficient for the linear equations for the seven compounds lay between 0.9978 and 0.9992. The limits of detection and quantification lay in the range of 0.044-0.084 and 0.13-0.25 microg/ml, respectively. The relative recovery rates for the seven compounds lay between 96.63+/-2.43 and 103.55+/-2.77%. Coefficient variation for intra-day and inter-day precisions lay in the range of 0.20-1.84 and 0.28-1.86%, respectively. Based upon our validation results, this analytical technique is a convenient method to simultaneous quantify numerous bioactive compounds derived from licorice, featuring good quantification parameters, accuracy and precision.

[Study on the fingerprint of Morus alba from different habitats by HPLC].

PubMed

Chen, Cheng; Li, Hong-Bo; Wang, Liu-Ping; Li, Yun-Rong; Xin, Ning

2012-12-01

To establish HPLC fingerprint of Morus alba from different habitats by HPLC and provide basis for its quality control. HPLC analysis was performed on an Agilent XDB C18 Column (4.6 mm x 250 mm, 5 microm), gradient eluted composed of acetonitrile and 0.3% phosphate acid. The column temperature was set at 35 degrees C and the flow rate was 0.5 mL/min. The detective wavelength was 290 nm. The HPLC fingerprint for 10 batches of Morus alba was studied on their similarity. There were twelve common peaks in the fingerprint. The similarity of 7 batches was above 0.9 and the other batches had low similarity. The HPLC fingerprint can be used for quality control of Morus alba with high characteristics and specificity.

Evaluation of differences between dual salt-pH gradient elution and mono gradient elution using a thermodynamic model: Simultaneous separation of six monoclonal antibody charge and size variants on preparative-scale ion exchange chromatographic resin.

PubMed

Lee, Yi Feng; Jöhnck, Matthias; Frech, Christian

2018-02-21

The efficiencies of mono gradient elution and dual salt-pH gradient elution for separation of six mAb charge and size variants on a preparative-scale ion exchange chromatographic resin are compared in this study. Results showed that opposite dual salt-pH gradient elution with increasing pH gradient and simultaneously decreasing salt gradient is best suited for the separation of these mAb charge and size variants on Eshmuno ® CPX. Besides giving high binding capacity, this type of opposite dual salt-pH gradient also provides better resolved mAb variant peaks and lower conductivity in the elution pools compared to single pH or salt gradients. To have a mechanistic understanding of the differences in mAb variants retention behaviors of mono pH gradient, parallel dual salt-pH gradient, and opposite dual salt-pH gradient, a linear gradient elution model was used. After determining the model parameters using the linear gradient elution model, 2D plots were used to show the pH and salt dependencies of the reciprocals of distribution coefficient, equilibrium constant, and effective ionic capacity of the mAb variants in these gradient elution systems. Comparison of the 2D plots indicated that the advantage of opposite dual salt-pH gradient system with increasing pH gradient and simultaneously decreasing salt gradient is the noncontinuous increased acceleration of protein migration. Furthermore, the fitted model parameters can be used for the prediction and optimization of mAb variants separation in dual salt-pH gradient and step elution. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Reverse-phase HPLC analysis of human alpha crystallin.

PubMed

Swamy, M S; Abraham, E C

1991-03-01

A rapid and highly sensitive reverse-phase HPLC (RP-HPLC) method was used to separate crystallin subunits from human alpha crystallin. Three distinct peaks were separated; by electrophoretic and immunological analyses the first and second peaks were identified as alpha B and alpha A respectively. On the other hand, peak 3 appeared to be a modified form of alpha crystallin. The ratio of alpha A and alpha B proteins was 3:1 in 1 day old lenses which gradually changed to 2:1 in 17 year old lenses and to 1:1 in the 50 and 82 year old whole lenses and 82 year old lens cortex, with a concomitant increase in the modified alpha, suggesting that alpha A subunits are relatively more involved in aggregation. Analysis of the 82 year old lens nucleus also supported this conclusion. The RP-HPLC analysis of the HMW aggregate fraction showed substantial enrichment of the modified alpha. The alpha A and alpha B subunits independently reassociated to form polymeric alpha crystallin whereas the modified alpha reassociated to form HMW aggregates as shown by molecular sieve HPLC. Hence it appears that the HMW aggregate peak was constituted by modified alpha crystallin. Only in the peak 3 material the 280 nm absorbance was about 2-fold higher than what was expected from the actual protein content. The data suggest that the changes induced by post-translational modifications may have some role in the formation of modified alpha. The present RP-HPLC method is useful in separating these modified alpha from the unmodified alpha A and alpha B subunits.

Comparative studies on performance of CCC and preparative RP-HPLC in separation and purification of steroid saponins from Dioscorea zingiberensis C.H.Wright

PubMed Central

Zhang, Xinxin; Liang, Jinru; Zhang, Yongmin; Liu, Jianli; Sun, Wenji; Ito, Yoichiro

2015-01-01

Steroid saponins from Dioscorea zingiberensis C.H.Wright were separated for the first time using two chromatographic methods for comparison: counter-current chromatography (CCC) coupled with evaporative light scattering detector (ELSD) and preparative reversed phase high-performance liquid chromatography (RP-HPLC) with an ultraviolet detector. Ethyl acetate-n-butanol-methanol-water (4:1:2:4, v/v) was chosen as the two-phase solvent system for CCC, while the acetonitrile-water (25:75 for the first step and15:85 for the second step, v/v) was used as the mobile phase in the preparative RP-HPLC. The following five steroid saponins were purified by theses two chromatographic methods, in one-step operation by CCC and by two-step operation in preparative RP-HPLC: 1) 26-O-β-D- glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 26-triol-3-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (compound A), 2) 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 4) 26-triol-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (compound B), 3) 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 26-triol-3-O-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside (compound C), 4) 26-O-β-D-glucopyranosyl-(25R)-furost-5, 20(22)-diene-3β, 26-diol-3-O-{α-L-rhamnopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→2)]}-β-D-glucopyranoside (compound D) and 5) 26-O-β-D-glucopyranosyl-(25R)-furost-5, 20(22)-diene-3β, 26-diol-3-O-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosy-(1→2)]-β-D-glucopyranoside (compound E). The purities of these five steroid saponins separated by both methods were over 95%, and structural identification of these compounds was performed by ESI-MS, and 13C NMR. Comparison of these two established approaches revealed that CCC required a longer separation time but with less solvent consumption, whereas preparative RP-HPLC gave a shorter separation time but

Determination and validation of six sunscreen agents in suncare products by UPLC and HPLC.

PubMed

Lee, So-Mi; Jeong, Hye-Jin; Chang, Ih Seop

2008-01-01

Methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine are sunscreen agents that have hydrophobic behaviors in common. They were not normally assayed with the following four sunscreen agents that have hydrophilic behaviors in a single chromatographic run: ethylhexyl methoxycinnamate, isoamyl p-methoxycinnamate, ethylhexyl salicylate, and ethylhexyl triazone. For that reason, methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine require much time in order to assay products with those materials. A rapid, selective, and reproducible determination method needs to be developed for the simultaneous examination of methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine with the sunscreen agents, ethylhexyl methoxycinnamate, isoamyl p-methoxycinnamate, ethylhexyl salicylate, and ethylhexyl triazone. This new technique could reduce time in examining the sunscreen agents and be effective for quality control of suncare products. In this paper, the HPLC and UPLC system is used for developing the determination of the sunscreen agents. Several evaluations of some mixtures of eluents and columns were obtained for the optimal condition of separation. In HPLC, the optimal peak resolution was obtained through ethanol-water gradient elution and a 75-mm C18 column with a 3.5-microm-sized particle on a flow rate of 1.0 ml/min. In UPLC, the most distinctive peak resolution was obtained through methanol-water gradient elution and a 50-mm C18 column with a 1.7-microm-sized particle on a flow rate 0.4 ml/min. Both of those chromatographic determination methods could be used in the examination of six types of sunscreen agents without any interference from other product excipients in the agents. The proposed determination methods were validated for specificity, linearity, repeatability, system stability, intermediate precision, and accuracy

[HPLC fingerprint analysis of flavonoids of phyllanthi fructus from different habitats].

PubMed

Wang, Fei; Wang, Shuai; Meng, Xian-sheng; Bao, Yong-rui; Zhu, Ying-huan

2014-11-01

To establish the HPLC fingerprint of flavonoids of Phyllanthi Fructus from different habitats. HPLC method was adopted. The flavonoids composition of Phyllanthi Fructus from 10 different habitats was determined on an Agilent C, chromatographic column with 0. 5% formic acid water (A)-acetonitrile (B) as the mobile phase in gradient elution under the wavelength of 254 nm. The HPLC fingerprints of flavonoids composition of Phyllanthi Fructus were established to evaluate the qualitiy of them. The HPLC fingerprints of flavonoids composition of Phyllanthi Fructus from 10 different habitats were established. 18 common peaks were found and the similarities of them were more than 0. 90 except the ones from Guangxi and Guangdong. The method is simple, accurate and repeatable. It can be used for research and quality control of the effective components in Phyllanthi Fructus.

Two-dimensional chromatography of complex polymers, 8. Separation of fatty alcohol ethoxylates simultaneously by end group and chain length.

PubMed

Raust, Jacques-Antoine; Bruell, Adele; Sinha, Pritish; Hiller, Wolf; Pasch, Harald

2010-09-01

A comprehensive two-dimensional liquid chromatography system was developed to precisely describe the molecular heterogeneity of fatty alcohol ethoxylates. The end-group functionality was analyzed by gradient HPLC while ethylene oxide oligomer distributions were characterized by liquid adsorption chromatography. A baseline separation of all functionality fractions irrespective of the ethylene oxide oligomer chain length was achieved on nonpolar X-Terra(®) C(18) with a methanol-water gradient, whereas an isocratic flow of isopropanol-water on a polar Chromolith(®) Si column gave a separation according to the oligomer chain length without interference of the end-group distribution. The combination of these two methods to conduct online two-dimensional liquid chromatography experiments resulted in a comprehensive two-dimensional picture on the molecular heterogeneity of the sample.

Application and Prospect of Superconducting High Gradient Magnetic Separation in Disposal of Micro-fine Tailings

NASA Astrophysics Data System (ADS)

Yang, Changqiao; Li, Suqin; Guo, Zijie; Kong, Jiawei

2017-12-01

Magnetic separation technology is playing an increasingly important role in the field of environmental protection such as waste gas, waste water and solid waste treatment. As a new type of solid waste treatment technology, superconducting high gradient magnetic separation (HGMS) is mainly applied in the separation of micro-fine weakly magnetic particles because of the advantages of high separation efficiency, energy saving, simple equipment and easy automation. In this paper, the basic principle of superconducting HGMS was firstly introduced, then the research status of scholars at home and aboard on the disposal of micro-fine tailings were summarized. Finally, the direction of development for HGMS was put forward.

Simultaneous separation and determination of 15 organic UV filters in sunscreen cosmetics by HPLC-ESI-MS/MS.

PubMed

Meng, X; Ma, Q; Bai, H; Wang, Z; Han, C; Wang, C

2017-08-01

A comprehensive methodology for the simultaneous determination of 15 multiclass organic UV filters in sunscreen cosmetics was developed using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Sunscreen cosmetics of various matrices, such as toning lotion, emulsion, cream and lipstick, were analysed. Ultrasound-assisted extraction (UAE) was utilized as the extraction technique for sample preparation. The 15 UV filters were chromatographically separated by two groups of mobile phase system on an XBridge C 18 analytical column (150 × 2.1 mm I.D., 3.5 μm particle size) and quantified using HPLC-ESI-MS/MS. The quantitation was performed using the external calibration method. The established method was validated in terms of linearity, sensitivity, specificity, accuracy, stability, intraday and interday precisions, recovery and matrix effect. The method was also applied for the determination of UV filters in commercial sunscreen cosmetics. The experimental results demonstrated that the developed method was accurate, rapid and sensitive and can be used for the analytical control of sunscreen cosmetics. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

[HPLC fingerprint of Calendula officinalis flower].

PubMed

Xing, Zhan-Fen; Cheng, Hong-Da; Zhang, Ping-Ping; Gong, Lei; Ma, Li-Ya

2014-07-01

To establish an HPLC fingerprint of Calendula officinalis flower for its quality control. Hypersil ODS C18 column (250 mm x 4.6 mm, 5 μm) was used with acetonitrile and water as mobile phase in a gradient mode at the flow rate of 1.0 mL/min. The detection wavelength was 220 nm and the temperature of column was set at 35 degrees C. The similarity was analyzed with the Estimating System of Similarity on the Chinese Medicine Fingerprint Chromatogram. The HPLC fingerprint of Calendula officinalis flower containing eleven peaks was set up. The similarity of Calendula officinalis flower from different habitats was greater than 0.90. This method is easy and reliable, which can be used to judge the habitat and control the quality of Calendula officinalis flower.

Quantitative determination of limonin in citrus juices by HPLC using computerized solvent optimization.

PubMed

Shaw, P E; Wilson, C W

1988-09-01

The commercially available computer program, Drylab, for optimization of separations by high-performance liquid chromatography (HPLC) using binary solvent mixtures is used to improve an HPLC method for separation of the bitter principle, limonin, in grapefruit and navel orange juices. Best conditions for separation of limonin in a reasonable time are 30 to 32% acetonitrile in water at 0.9 mL/min using a 5-micron C18 column 10 cm long. These conditions are used to analyze grapefruit and navel orange juice samples, and these HPLC results are compared with values determined by enzyme immunoassay or thin-layer chromatography (TLC) on the same samples.

Blind separation of positive sources by globally convergent gradient search.

PubMed

Oja, Erkki; Plumbley, Mark

2004-09-01

The instantaneous noise-free linear mixing model in independent component analysis is largely a solved problem under the usual assumption of independent nongaussian sources and full column rank mixing matrix. However, with some prior information on the sources, like positivity, new analysis and perhaps simplified solution methods may yet become possible. In this letter, we consider the task of independent component analysis when the independent sources are known to be nonnegative and well grounded, which means that they have a nonzero pdf in the region of zero. It can be shown that in this case, the solution method is basically very simple: an orthogonal rotation of the whitened observation vector into nonnegative outputs will give a positive permutation of the original sources. We propose a cost function whose minimum coincides with nonnegativity and derive the gradient algorithm under the whitening constraint, under which the separating matrix is orthogonal. We further prove that in the Stiefel manifold of orthogonal matrices, the cost function is a Lyapunov function for the matrix gradient flow, implying global convergence. Thus, this algorithm is guaranteed to find the nonnegative well-grounded independent sources. The analysis is complemented by a numerical simulation, which illustrates the algorithm.

Combining Step Gradients and Linear Gradients in Density.

PubMed

Kumar, Ashok A; Walz, Jenna A; Gonidec, Mathieu; Mace, Charles R; Whitesides, George M

2015-06-16

Combining aqueous multiphase systems (AMPS) and magnetic levitation (MagLev) provides a method to produce hybrid gradients in apparent density. AMPS—solutions of different polymers, salts, or surfactants that spontaneously separate into immiscible but predominantly aqueous phases—offer thermodynamically stable steps in density that can be tuned by the concentration of solutes. MagLev—the levitation of diamagnetic objects in a paramagnetic fluid within a magnetic field gradient—can be arranged to provide a near-linear gradient in effective density where the height of a levitating object above the surface of the magnet corresponds to its density; the strength of the gradient in effective density can be tuned by the choice of paramagnetic salt and its concentrations and by the strength and gradient in the magnetic field. Including paramagnetic salts (e.g., MnSO4 or MnCl2) in AMPS, and placing them in a magnetic field gradient, enables their use as media for MagLev. The potential to create large steps in density with AMPS allows separations of objects across a range of densities. The gradients produced by MagLev provide resolution over a continuous range of densities. By combining these approaches, mixtures of objects with large differences in density can be separated and analyzed simultaneously. Using MagLev to add an effective gradient in density also enables tuning the range of densities captured at an interface of an AMPS by simply changing the position of the container in the magnetic field. Further, by creating AMPS in which phases have different concentrations of paramagnetic ions, the phases can provide different resolutions in density. These results suggest that combining steps in density with gradients in density can enable new classes of separations based on density.

Feasibility of turbidity removal by high-gradient superconducting magnetic separation.

PubMed

Zeng, Hua; Li, Yiran; Xu, Fengyu; Jiang, Hao; Zhang, Weimin

2015-01-01

Several studies have focused on pollutant removal by magnetic seeding and high-gradient superconducting magnetic separation (HGSMS). However, few works reported the application of HGSMS for treating non-magnetic pollutants by an industrial large-scale system. The feasibility of turbidity removal by a 600 mm bore superconducting magnetic separation system was evaluated in this study. The processing parameters were evaluated by using a 102 mm bore superconducting magnetic separation system that was equipped with the same magnetic separation chamber that was used in the 600 mm bore system. The double-canister system was used to process water pollutants. Analytical grade magnetite was used as a magnetic seed and the turbidity of the simulated raw water was approximately 110 NTU, and the effects of polyaluminum chloride (PAC) and magnetic seeds on turbidity removal were evaluated. The use of more PAC and magnetic seeds had few advantages for the HGSMS at doses greater than 8 and 50 mg/l, respectively. A magnetic intensity of 5.0 T was beneficial for HGSMS, and increasing the flow rate through the steel wool matrix decreased the turbidity removal efficiency. In the breakthrough experiments, 90% of the turbidity was removed when 100 column volumes were not reached. The processing capacity of the 600 mm bore industry-scale superconducting magnetic separator for turbidity treatment was approximately 78.0 m(3)/h or 65.5 × 10(4) m(3)/a. The processing cost per ton of water for the 600 mm bore system was 0.1 $/t. Thus, the HGSMS separator could be used in the following special circumstances: (1) when adequate space is not available for traditional water treatment equipment, especially the sedimentation tank, and (2) when decentralized sewage treatment HGSMS systems are easier to transport and install.

Fingerprint of Hedyotis diffusa Willd. by HPLC-MS.

PubMed

Yang, Ting; Yang, Yi-Hua; Yang, Ju-Yun; Chen, Ben-Mei; Duan, Ju-Ping; Yu, Shu-Yi; Ouyang, Hong-Tao; Cheng, Jun-Ping; Chen, Yu-Xiang

2008-01-01

A HPLC-MS fingerprint method has been developed based on the consistent chromatographic features of the major chemical constituents among 10 batches of Hedyotis diffusa Willd. Chromatographic separation was conducted on a Hypersil-Keystone Hypurity C(18) column using methanol:water:acetic acid as the mobile phase. Major compounds, including oleanolic acid, ursolic acid and ferulic acid, were analysed by HPLC-MS. Their analysis was ascertained by comparison with data derived from the standard compounds. The HPLC-MS fingerprint was successfully applied to analyse and differentiate samples from different geographical origins, or processing methods. H. diffusa was well distinguished from Hedyotis chrysotricha by HPLC-MS. Therefore the establishment of fingerprint of H. diffusa is critical in assessing and controlling its overall quality.

Selenium speciation analysis of Misgurnus anguillicaudatus selenoprotein by HPLC-ICP-MS and HPLC-ESI-MS/MS

USDA-ARS?s Scientific Manuscript database

Analytical methods for selenium (Se) speciation were developed using high performance liquid chromatography (HPLC) coupled to either inductively coupled plasma mass spectrometry (ICP-MS) or electrospray ionization tandem mass spectrometry (ESI-MS/MS). Separations of selenomethionine (Se-Met) and sel...

Thin-layer chromatography with stationary phase gradient as a method for separation of water-soluble vitamins.

PubMed

Cimpoiu, Claudia; Hosu, Anamaria; Puscas, Anitta

2012-02-03

The group of hydrophilic vitamins play an important role in human health, and their lack or excess produces specific diseases. Therefore, the analysis of these compounds is indispensable for monitoring their content in pharmaceuticals and food in order to prevent some human diseases. TLC was successfully applied in the analysis of hydrophilic vitamins, but the most difficult problem in the simultaneous analysis of all these compounds is to find an optimum stationary phase-mobile phase system due to different chemical characteristics of analytes. Unfortunately structural analogues are difficult to separate in one chromatographic run, and this is the case in hydrophilic vitamins investigations. TLC gives the possibility to perform two-dimensional separations by using stationary phase gradient achieving the highest resolution by combining two systems with different selectivity. The goal of this work was to develop a method of analysis enabling separation of hydrophilic vitamins using TLC with adsorbent gradient. The developed method was used for identifying the water-soluble vitamins in alcoholic extracts of Hippophae rhamnoides and of Ribes nigrum. Copyright © 2011 Elsevier B.V. All rights reserved.

Simultaneous determination of chlorogenic acid, caffeic acid, alantolactone and isoalantolactone in Inula helenium by HPLC.

PubMed

Wang, Jin; Zhao, Yong-ming; Zhang, Man-li; Shi, Qing-wen

2015-04-01

A rapid and sensitive high-performance liquid chromatographic (HPLC) method was developed for the simultaneous separation and determination of chlorogenic acid, caffeic acid, alantolactone and isoalantolactone in Inula helenium. The HPLC separation was performed on an Elite Hypersil C18 column (200 × 4.6 mm i.d., 5 µm particle size) with a gradient elution of solvent A (acetonitrile) and solvent B (0.1% phosphoric acid in water) at a flow rate of 1.0 mL/min. Detection was monitored at 225 nm. The recovery of chlorogenic acid ranged from 95.6 to 107.7%, the recovery of caffeic acid ranged from 95.4 to 104.2%, the recovery of alantolactone ranged from 95.8 to 100.8% and the recovery of isoalantolactone ranged from 96.5 to 102.3%. The retention times for chlorogenic acid, caffeic acid, alantolactone and isoalantolactone were 5.2, 7.1, 25.6 and 26.6 min with the limits of detection of 0.069, 0.021, 0.039 and 0.051 µg/mL, respectively. Relative standard deviation for the intra-day and inter-day was ≤2.5%. The validated method is reliable for the routine control of these four compounds in I. helenium. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Simultaneous qualification and quantification of baccharane glycosides in Impatientis Semen by HPLC-ESI-MSD and HPLC-ELSD.

PubMed

Li, Hui-Jun; Yu, Jun-Jie; Li, Ping

2011-03-25

This study presents a high performance liquid chromatography (HPLC) with electrospray ionization mass spectrometric detection (ESI-MSD) and evaporative light scattering detection (ELSD) method for the simultaneous qualification and quantification of eight major baccharane glycosides, namely hosenlosides A, B, C, F, G, K, L, and M in Impatientis Semen, a Chinese herbal medicine derived from the seeds of Impatiens balsamina L. In order to achieve optimum performance, several extraction parameters (including extraction solvent, extraction mode, extraction time) were optimized. The baccharane glycosides were separated on a Shim-pack CLC-ODS column with gradient elution of water and methanol. Temperature for the ELSD drift tube was set at 98°C and the nitrogen flow rate was 2.7l/min. The unambiguous identities of the analytes were realized by comparing retention times and mass data with those of reference compounds. The developed method was fully validated in terms of linearity, sensitivity, precision, repeatability, recovery as well as robustness, and subsequently applied to evaluate the quality of 14 batches of Impatientis Semen commercial samples from different collections. Copyright © 2010 Elsevier B.V. All rights reserved.

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from Escherichia coli populations?

PubMed

Arana, Inés; Orruño, Maite; Seco, Carolina; Muela, Alicia; Barcina, Isabel

2008-03-01

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.

Application of an efficient strategy based on liquid-liquid extraction, high-speed counter-current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum.

PubMed

Chen, Tao; Liu, Yongling; Zou, Denglang; Chen, Chen; You, Jinmao; Zhou, Guoying; Sun, Jing; Li, Yulin

2014-01-01

This study presents an efficient strategy based on liquid-liquid extraction, high-speed counter-current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. A new solvent system composed of petroleum ether/ethyl acetate/water (4:2:1, v/v/v) was developed for the liquid-liquid extraction of the crude extract from R. tanguticum. As a result, emodin, aloe-emodin, physcion, and chrysophanol were greatly enriched in the organic layer. In addition, an efficient method was successfully established to separate and purify the above anthraquinones by high-speed counter-current chromatography and preparative HPLC. This study supplies a new alternative method for the rapid enrichment, separation, and purification of emodin, aloe-emodin, physcione, and chrysophanol. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HPLC-UV Method for the Identification and Screening of Hydroquinone, Ethers of Hydroquinone and Corticosteroids Possibly Used as Skin-Whitening Agents in Illicit Cosmetic Products.

PubMed

Gimeno, Pascal; Maggio, Annie-Françoise; Bancilhon, Marjorie; Lassu, Nelly; Gornes, Hervé; Brenier, Charlotte; Lempereur, Laurent

2016-03-01

Corticosteroids, hydroquinone and its ethers are regulated in cosmetics by the Regulation 1223/2009. As corticosteroids are forbidden to be used in cosmetics and cannot be present as contaminants or impurities, an identification of one of these illicit compounds deliberately introduced in these types of cosmetics is enough for market survey control. In order to quickly identify skin-whitening agents present in illegal cosmetics, this article proposes an HPLC-UV method for the identification and screening of hydroquinone, 3 ethers of hydroquinone and 39 corticosteroids that may be found in skin-whitening products. Two elution gradients were developed to separate all compounds. The main solvent gradient (A) allows the separation of 39 compounds among the 43 compounds considered in 50 min. Limits of detection on skin-whitening cosmetics are given. For compounds not separated, a complementary gradient elution (B) using the same solvents is proposed. Between 2004 and 2009, a market survey on "skin-whitening cosmetic" was performed on 150 samples and highlights that more than half of the products tested do not comply with the Cosmetic Regulation 1223/2009 (amending the Council Directive 76/768/EEC). © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Validation of HPLC method for the simultaneous and quantitative determination of 12 UV-filters in cosmetics.

PubMed

Nyeborg, M; Pissavini, M; Lemasson, Y; Doucet, O

2010-02-01

The aim of the study was the validation of a high-performance liquid chromatography (HPLC) method for the simultaneous and quantitative determination of twelve commonly used organic UV-filters (phenylbenzimidazole sulfonic acid, benzophenone-3, isoamyl p-methoxycinnamate, diethylamino hydroxybenzoyl hexyl benzoate, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, butyl methoxydibenzoylmethane, diethylhexyl butamido triazone, ethylhexyl triazone, methylene bis-benzotriazolyl tetramethylbutylphenol and bis-ethylhexyloxyphenol methoxyphenyl triazine) contained in suncare products. The separation and quantitative determination was performed in <30 min, using a Symmetry Shield(R) C18 (5 microm) column from Waters and a mobile phase (gradient mode) consisting of ethanol and acidified water. UV measurements were carried out at multi-wavelengths, according to the absorption of the analytes.

Microfluidic device having an immobilized pH gradient and PAGE gels for protein separation and analysis

DOEpatents

Sommer, Gregory J.; Hatch, Anson V.; Singh, Anup K.; Wang, Ying-Chih

2012-12-11

Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

Microfluidic device having an immobilized pH gradient and page gels for protein separation and analysis

DOEpatents

Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

2014-05-20

Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

Development of superconducting high gradient magnetic separation system for highly viscous fluid for practical use

NASA Astrophysics Data System (ADS)

Hayashi, S.; Mishima, F.; Akiyama, Y.; Nishijima, S.

2011-11-01

In the industrial plants processing highly viscous fluid such as foods or industrial products, it is necessary to remove the metallic wear debris originating from pipe in manufacturing line which triggers quality loss. In this study, we developed a high gradient magnetic separation (HGMS) system which consists of superconducting magnet to remove the metallic wear debris. The magnetic separation experiment and the particle trajectory simulation were conducted with polyvinyl alcohol (PVA) as a model material (viscosity coefficient was 10 Pa s, which is 10,000 times higher than that in water). In order to develop a magnetic separation system for practical use, the particle trajectory simulation by using solenoidal superconducting magnet was conducted, and the possibility of the magnetic separation for removing ferromagnetic stainless steel (SUS) particles in highly viscous fluid of 10 Pa s was indicated. Based on the results, the number of filters to obtain required separation efficiency was examined to design the practical separation system.

Development and application of a validated HPLC method for the analysis of dissolution samples of levothyroxine sodium drug products

PubMed Central

Collier, J.W.; Shah, R.B.; Bryant, A.R.; Habib, M.J.; Khan, M.A.; Faustino, P.J.

2011-01-01

A rapid, selective, and sensitive gradient HPLC method was developed for the analysis of dissolution samples of levothyroxine sodium tablets. Current USP methodology for levothyroxine (l-T4) was not adequate to resolve co-elutants from a variety of levothyroxine drug product formulations. The USP method for analyzing dissolution samples of the drug product has shown significant intra- and inter-day variability. The sources of method variability include chromatographic interferences introduced by the dissolution media and the formulation excipients. In the present work, chromatographic separation of levothyroxine was achieved on an Agilent 1100 Series HPLC with a Waters Nova-pak column (250mm × 3.9mm) using a 0.01 M phosphate buffer (pH 3.0)–methanol (55:45, v/v) in a gradient elution mobile phase at a flow rate of 1.0 mL/min and detection UV wavelength of 225 nm. The injection volume was 800 µL and the column temperature was maintained at 28 °C. The method was validated according to USP Category I requirements. The validation characteristics included accuracy, precision, specificity, linearity, and analytical range. The standard curve was found to have a linear relationship (r2 > 0.99) over the analytical range of 0.08–0.8 µg/mL. Accuracy ranged from 90 to 110% for low quality control (QC) standards and 95 to 105% for medium and high QC standards. Precision was <2% at all QC levels. The method was found to be accurate, precise, selective, and linear for l-T4 over the analytical range. The HPLC method was successfully applied to the analysis of dissolution samples of marketed levothyroxine sodium tablets. PMID:20947276

Semi-Preparative Separation of 10 Caffeoylquinic Acid Derivatives Using High Speed Counter-Current Chromatogaphy Combined with Semi-Preparative HPLC from the Roots of Burdock (Arctium lappa L.).

PubMed

Zheng, Zhenjia; Wang, Xiao; Liu, Pengli; Li, Meng; Dong, Hongjing; Qiao, Xuguang

2018-02-15

Burdock roots are healthy dietary supplements and a kind of famous traditional Chinese medicine, which contains large amounts of caffeoylquinic acid derivatives. However, little research has been reported on the preparative separation of these compounds from burdock roots. In the present study, a combinative method of HSCCC and semi-preparative HPLC was developed for the semi-preparative separation of caffeoylquinic acid derivatives from the burdock roots. The ethyl acetate extract of burdock roots was first fractionated by MCI macroporous resin chromatography and give three fractions (Fr. 1-3) from the elution of 40% methanol. Then, these three fractions (120 mg) were separately subjected to HSCCC for purification with the solvent system composed of petroleum ether-ethyl acetate-methanol-water at different volume ratios, and the mixtures were further purified by semi-preparative HPLC. As a result, a total of eight known caffeoylquinic acid derivatives including 3- O -caffeoylquinic acid (32.7 mg, 95.7%), 1,5- O - dicaffeoylquinic acid (4.3 mg, 97.2%), 3- O -caffeoylquinic acid methyl ester (12.1 mg, 93.2%), 1,3- O -dicaffeoylquinic acid (42.9 mg, 91.1%), 1,5- O -dicaffeoyl-3- O -(4-maloyl)-quinic acid (4.3 mg, 84.5%), 4,5- O -dicaffeoylquinic acid (5.3 mg, 95.5%), 1,5- O -dicaffeoyl-3- O -succinylquinic acid (8.7 mg, 93.4%), and 1,5- O -dicaffeoyl-4- O -succinylquinic acid (1.7 mg, 91.8%), and two new compounds were obtained. The new compounds were 1,4- O -dicaffeoyl-3-succinyl methyl ester quinic acid (14.6 mg, 96.1%) and 1,5- O -dicaffeoyl-3- O -succinyl methyl ester quinic acid (3.1 mg, 92.6%), respectively. The research indicated that the combination of HSCCC and semi-preparative HPLC is a highly efficient approach for preparative separation of the instability and bioactive caffeoylquinic acid derivatives from natural products.

[HPLC fingerprint of the antiarrhythmic fraction of Valeriana officinalis].

PubMed

Duan, Xue-Yun; Gong, Zhan-Feng; Chen, Shu-He; Fang, Ying; Liu, Yan-Wen

2009-06-01

To establish HPLC fingerprints of the Antiarrhythmic fraction of Valeriana officinalis. Agilent C18 (250 mm x 4.6 mm, 5 microm) column was used and the acetonitrile-water was chosen as the mobile phase in a gradient mode. The column temperature was 380 degrees C and the detection wavelength was 218 nm. The detection time was 70 min, and the flow rate was 1.0 mL/ min. Fifteen characteristic peaks were indicated in HPLC fingerprints. The relative retention time and the ranges of relative areas of the common peaks were also determined. This method is simple and accurate with a good reproducibility and provides a reference standard for the quality control of Valeriana officinalis.

Quantification of Aconitum alkaloids in aconite roots by a modified RP-HPLC method.

PubMed

Jiang, Zhi-Hong; Xie, Ying; Zhou, Hua; Wang, Jing-Rong; Liu, Zhong-Qiu; Wong, Yuen-Fan; Cai, Xiong; Xu, Hong-Xi; Liu, Liang

2005-01-01

The three Aconitum alkaloids, aconitine (1), mesaconitine (2) and hypaconitine (3), are pharmacologically active but also highly toxic. A standardised method is needed for assessing the levels of these alkaloids in aconite roots in order to ensure the safe use of these plant materials as medicinal herbs. By optimising extraction, separation and measurement conditions, a reliable, reproducible and accurate method for the quantitative determination of all three Aconitum alkaloids in unprocessed and processed aconite roots has been developed. This method should be appropriate for use in the quality control of Aconitum products. The three Aconitum alkaloids were separated by a modified HPLC method employing a C18 column gradient eluted with acetonitrile and ammonium bicarbonate buffer. Quantification of Aconitum alkaloids, detected at 240 nm, in different batches of samples showed that the content of 1, 2 and 3 varied significantly. In general, the alkaloid content of unprocessed roots was higher than that of processed roots. These variations were considered to be the result of differences in species, processing methods and places of origin of the samples.

Rational and Efficient Preparative Isolation of Natural Products by MPLC-UV-ELSD based on HPLC to MPLC Gradient Transfer.

PubMed

Challal, Soura; Queiroz, Emerson Ferreira; Debrus, Benjamin; Kloeti, Werner; Guillarme, Davy; Gupta, Mahabir Prashad; Wolfender, Jean-Luc

2015-11-01

In natural product research, the isolation of biomarkers or bioactive compounds from complex natural extracts represents an essential step for de novo identification and bioactivity assessment. When pure natural products have to be obtained in milligram quantities, the chromatographic steps are generally labourious and time-consuming. In this respect, an efficient method has been developed for the reversed-phase gradient transfer from high-performance liquid chromatography to medium-performance liquid chromatography for the isolation of pure natural products at the level of tens of milligrams from complex crude natural extracts. The proposed method provides a rational way to predict retention behaviour and resolution at the analytical scale prior to medium-performance liquid chromatography, and guarantees similar performances at both analytical and preparative scales. The optimisation of the high-performance liquid chromatography separation and system characterisation allows for the prediction of the gradient at the medium-performance liquid chromatography scale by using identical stationary phase chemistries. The samples were introduced in medium-performance liquid chromatography using a pressure-resistant aluminium dry load cell especially designed for this study to allow high sample loading while maintaining a maximum achievable flow rate for the separation. The method has been validated with a mixture of eight natural product standards. Ultraviolet and evaporative light scattering detections were used in parallel for a comprehensive monitoring. In addition, post-chromatographic mass spectrometry detection was provided by high-throughput ultrahigh-performance liquid chromatography time-of-flight mass spectrometry analyses of all fractions. The processing of all liquid chromatography-mass spectrometry data in the form of an medium-performance liquid chromatography x ultra high-performance liquid chromatography time-of-flight mass spectrometry matrix enabled an

On the feasibility to conduct gradient liquid chromatography separations in narrow-bore columns at pressures up to 2000bar.

PubMed

De Pauw, Ruben; Swier, Tim; Degreef, Bart; Desmet, Gert; Broeckhoven, Ken

2016-11-18

The limits in operating pressures are extended for narrow-bore columns in gradient elution up to 2000bar. As the required pumps for these pressures are incompatible with common chromatographic solvents and are not suitable to apply a mobile phase composition gradient, a mobile phase delivery and injection system is described and experimentally validated which allows to use any possible chromatographic solvent in isocratic and gradient elution. The mobile phase delivery and injection system also allows to perform multiple separations without the need to depressurize the column. This system consists out of 5 dual on/off valves and two large volume loops in which the gradient and equilibration volume of initial mobile phase are loaded by a commercial liquid chromatography pump. The loops are then flushed toward the column at extreme pressures. The mobile phase delivery and injection system is first evaluated in isocratic elution and shows a comparable performance to a state-of-the-art commercial flow-through-needle injector but with twice the pressure rating. Distortion of the loaded gradient by dispersion in the gradient storage loop is studied. The effect of the most important parameters (such as flow rate, pressure and gradient steepness) is experimentally investigated. Different gradient steepnesses and volumes can be applied at different flow rates and operating pressures with a good repeatability. Due to the isobaric operation of the pumps, the gradient is monitored in real-time by a mass flow meter installed at the detector outlet. The chromatograms are then converted from time to volume-base. A separation of a 19-compound sample is performed on a 300×2.1mm column at 1000bar and on a 600×2.1mm column at 2000bar. The peak capacity was found to increase from 141 to 199 and thus scales with L as is predicted by theory. This allows to conclude that the inlet pressure for narrow-bore columns in gradient elution can be increased up to 2000bar without fundamental

CENTRIFUGAL SEPARATORS

DOEpatents

Skarstrom, C.

1959-03-10

A centrifugal separator is described for separating gaseous mixtures where the temperature gradients both longitudinally and radially of the centrifuge may be controlled effectively to produce a maximum separation of the process gases flowing through. Tbe invention provides for the balancing of increases and decreases in temperature in various zones of the centrifuge chamber as the result of compression and expansions respectively, of process gases and may be employed effectively both to neutralize harmful temperature gradients and to utilize beneficial temperaturc gradients within the centrifuge.

Quantification of the Triazole Antifungal Compounds Voriconazole and Posaconazole in Human Serum or Plasma Using Liquid Chromatography Electrospray Tandem Mass Spectrometry (HPLC-ESI-MS/MS).

PubMed

Molinelli, Alejandro R; Rose, Charles H

2016-01-01

Voriconazole and posaconazole are triazole antifungal compounds used in the treatment of fungal infections. Therapeutic drug monitoring of both compounds is recommended in order to guide drug dosing to achieve optimal blood concentrations. In this chapter we describe an HPLC-ESI-MS/MS method for the quantification of both compounds in human plasma or serum following a simple specimen preparation procedure. Specimen preparation consists of protein precipitation using methanol and acetonitrile followed by a cleanup step that involves filtration through a cellulose acetate membrane. The specimen is then injected into an HPLC-ESI-MS/MS equipped with a C18 column and separated over an acetonitrile gradient. Quantification of the drugs in the specimen is achieved by comparing the response of the unknown specimen to that of the calibrators in the standard curve using multiple reaction monitoring.

Multielemental speciation analysis by advanced hyphenated technique - HPLC/ICP-MS: A review.

PubMed

Marcinkowska, Monika; Barałkiewicz, Danuta

2016-12-01

Speciation analysis has become an invaluable tool in human health risk assessment, environmental monitoring or food quality control. Another step is to develop reliable multielemental speciation methodologies, to reduce costs, waste and time needed for the analysis. Separation and detection of species of several elements in a single analytical run can be accomplished by high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC/ICP-MS). Our review assembles articles concerning multielemental speciation determination of: As, Se, Cr, Sb, I, Br, Pb, Hg, V, Mo, Te, Tl, Cd and W in environmental, biological, food and clinical samples analyzed with HPLC/ICP-MS. It addresses the procedures in terms of following issues: sample collection and pretreatment, selection of optimal conditions for elements species separation by HPLC and determination using ICP-MS as well as metrological approach. The presented work is the first review article concerning multielemental speciation analysis by advanced hyphenated technique HPLC/ICP-MS. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection.

PubMed

Celik, Saliha Esin; Ozyürek, Mustafa; Güçlü, Kubilay; Apak, Reşat

2010-07-26

A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 microM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples. Copyright 2010 Elsevier B.V. All rights reserved.

Combining selection valve and mixing chamber for nanoflow gradient generation: Toward developing a liquid chromatography cartridge coupled with mass spectrometer for protein and peptide analysis.

PubMed

Chen, Apeng; Lu, Joann J; Gu, Congying; Zhang, Min; Lynch, Kyle B; Liu, Shaorong

2015-08-05

Toward developing a micro HPLC cartridge, we have recently built a high-pressure electroosmotic pump (EOP). However, we do not recommend people to use this pump to deliver an organic solvent directly, because it often makes the pump rate unstable. We have experimented several approaches to address this issue, but none of them are satisfactory. Here, we develop an innovative approach to address this issue. We first create an abruption (a dead-volume) within a fluid conduit. We then utilize an EOP to withdraw, via a selection valve, a train of eluent solutions having decreasing eluting power into the fluid conduit. When these solutions are further aspirated through the dead-volume, these solutions are partially mixed, smoothening concentration transitions between two adjacent eluent solutions. As these solutions are pushed back, through the dead-volume again, a smooth gradient profile is formed. In this work, we characterize this scheme for gradient formation, and we incorporate this approach with a high-pressure EOP, a nanoliter injection valve, and a capillary column, yielding a micro HPLC system. We then couple this micro HPLC with an electrospray ionization - mass spectrometer for peptide and protein separations and identifications. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly efficient peptide separations in proteomics. Part 2: bi- and multidimensional liquid-based separation techniques.

PubMed

Sandra, Koen; Moshir, Mahan; D'hondt, Filip; Tuytten, Robin; Verleysen, Katleen; Kas, Koen; François, Isabelle; Sandra, Pat

2009-04-15

Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.

Preparation of a poly(styrene-co-DPHA-co-EDMA) monolith and its application for the separation of small molecules and biomacromolecules by HPLC.

PubMed

Wang, Xixi; Li, Xueying; Jiang, Xiaoya; Dong, Peipei; Liu, Haiyan; Bai, Ligai; Yan, Hongyuan

2017-04-01

A high performance liquid chromatography (HPLC) monolithic column was prepared by redox polymerization of styrene, dipentaerythritol hexaacrylate (DPHA) and ethylene glycol dimethacrylate (EDMA) in a porogen system of n-propanol/PEG400. The monolith was characterized by scanning electron microscopy (SEM), mercury intrusion porosimetry (MIP) and the results indicated that the monolith had a stable and homogeneous structure. The porosity of the monolithic column was 75.86% and average pore diameter was 2.1µm. Several alkylbenzenes and anilines were used to evaluate the column performance in terms of hydrophobicity. Then the column was applied to separate small molecules including phytosterol and BSA tryptic digest. Finally, five standard proteins, egg white and plasma were separated respectively and high separation capacity of protein was obtained. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous determination of five marker constituents in Ssanghwa tang by HPLC/DAD

PubMed Central

Won, Jin Bae; Ma, Jin Yeul; Um, Young Ran; Ma, Choong Je

2010-01-01

A HPLC-DAD method was established for the simultaneous evaluation of five bioactive compounds in Ssanghwa tang (SHT) including glycyrrhizin, paeoniflorin, cinnamic acid, decursin and 6-gingerol. These compounds were separated in less than 40 min using a Dionex C18 column with a gradient elution system of water and methanol at a flow rate of 1 ml/min. Calibration curve of standard components presented excellent linear regression (R2 > 0.9903) within the test range. Limit of detection and limit of quantification varied from 0.07 to 0.46 μg/ml and 0.13 to 1.11 μg/ml, respectively. The relative standard deviations (RSDs) of data of the intraday and interday experiments were less than 3.67 and 5.73%, respectively. The accuracy of recovery test ranged from 95.98 to 105.88% with RSD values 0.10– 4.82%. PMID:20668576

Determination of Bortezomib in API Samples Using HPLC: Assessment of Enantiomeric and Diastereomeric Impurities.

PubMed

Kamalzadeh, Zahra; Babanezhad, Esmaeil; Ghaffari, Solmaz; Mohseni Ezhiyeh, Alireza; Mohammadnejad, Mahdieh; Naghibfar, Mehdi; Bararjanian, Morteza; Attar, Hossein

2017-08-01

A new, normal phase high performance liquid chromatography (NP-HPLC) method was developed for separation of Bortezomib (BZB) enantiomers and quantitative determination of (1S,2R)-enantiomer of BZB in active pharmaceutical ingredient (API) samples. The developed method was validated based on International Conference on Harmonisation (ICH) guidelines and it was proved to be accurate, precise and robust. The obtained resolution (RS) between the enantiomers was more than 2. The calibration curve for (1S,2R)-enantiomer was found to be linear in the concentration range of 0.24-5.36 mg/L with regression coefficient (R2) of 0.9998. Additionally, the limit of detection (LOD) and limit of quantification (LOQ) were 0.052 and 0.16 mg/L, respectively. Also, in this study, a precise, sensitive and robust gradient reversed-phase HPLC (RP-HPLC) method was developed and validated for determination of BZB in API samples. The detector response was linear over the concentration range of 0.26-1110.5 mg/L. The values of R2, LOD and LOQ were 0.9999, 0.084 and 0.25 mg/L, respectively. For both NP-HPLC and RP-HPLC methods, all of the RSD (%) values obtained in the precision study were <1.0%. System suitability parameters in terms of tailing factor (TF), number of theoretical plates (N) and RS were TF < 2.0, N > 2,000 and RS > 2.0. The performance of two common integration methods of valley to valley and drop perpendicular for drawing the baseline between two adjacent peaks were investigated for the determination of diastereomeric impurity (Imp-D) in the BZB-API samples. The results showed that the valley to valley method outperform the drop perpendicular method for calculation of Imp-D peak areas. Therefore, valley to valley method was chosen for peak integration. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Development and validation of reversed-phase HPLC gradient method for the estimation of efavirenz in plasma.

PubMed

Gupta, Shweta; Kesarla, Rajesh; Chotai, Narendra; Omri, Abdelwahab

2017-01-01

Efavirenz is an anti-viral agent of non-nucleoside reverse transcriptase inhibitor category used as a part of highly active retroviral therapy for the treatment of infections of human immune deficiency virus type-1. A simple, sensitive and rapid reversed-phase high performance liquid chromatographic gradient method was developed and validated for the determination of efavirenz in plasma. The method was developed with high performance liquid chromatography using Waters X-Terra Shield, RP18 50 x 4.6 mm, 3.5 μm column and a mobile phase consisting of phosphate buffer pH 3.5 and Acetonitrile. The elute was monitored with the UV-Visible detector at 260 nm with a flow rate of 1.5 mL/min. Tenofovir disoproxil fumarate was used as internal standard. The method was validated for linearity, precision, accuracy, specificity, robustness and data obtained were statistically analyzed. Calibration curve was found to be linear over the concentration range of 1-300 μg/mL. The retention times of efavirenz and tenofovir disoproxil fumarate (internal standard) were 5.941 min and 4.356 min respectively. The regression coefficient value was found to be 0.999. The limit of detection and the limit of quantification obtained were 0.03 and 0.1 μg/mL respectively. The developed HPLC method can be useful for quantitative pharmacokinetic parameters determination of efavirenz in plasma.

HPLC Determination of Esculin and Esculetin in Rat Plasma for Pharmacokinetic Studies.

PubMed

Rehman, Shaheed Ur; Kim, In Sook; Kang, Ki Sung; Yoo, Hye Hyun

2015-09-01

An optimized, sensitive and validated reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection is described for simultaneous determination of esculin and its aglycone, esculetin, in rat plasma. After addition of internal standard (chrysin), plasma samples were pretreated by solid-phase extraction and introduced into the HPLC system. Analytes were separated on a RP C18 column with a mobile phase of 0.075% acetic acid in water (solvent A) and 90% acetonitrile in solvent A (solvent B) using gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 338 nm. Calibration curves for esculin and esculetin were constructed over a range of 10-1,000 ng/mL. The developed method was found to be specific, precise and accurate. The method was successfully applied to study the pharmacokinetics of esculin and esculetin in rats. After oral administration of 120 mg/kg, the mean Cmax values were 340.3 and 316.5 ng/mL and the AUClast values were 377.3 and 1276.5 h ng/mL for esculin and esculetin, respectively. The bioavailability of esculin was calculated to be 0.62%. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Prediction of gradient retention data for hydrophilic interaction liquid chromatographic separation of native and fluorescently labeled oligosaccharides.

PubMed

Vaňková, Nikola; Česla, Petr

2017-02-17

In this work, we have investigated the predictive properties of mixed-mode retention model and oligomeric mixed-mode model, taking into account the contribution of monomeric units to the retention, in hydrophilic interaction liquid chromatography. The gradient retention times of native maltooligosaccharides and their fluorescent derivatives were predicted in the oligomeric series with number of monomeric glucose units in the range from two to seven. The maltooligosaccharides were separated on a packed column with carbamoyl-bonded silica stationary phase and 15 gradient profiles with different initial and final mobile phase composition were used with the gradient times 5; 7.5 and 10min. The predicted gradient retention times were compared for calculations based on isocratic retention data and gradient retention data, which provided better accuracy of the results. By comparing two different mobile phase additives, the more accurate retention times were predicted in mobile phases containing ammonium acetate. The acidic derivatives, prepared by reaction of an oligosaccharide with 2-aminobenzoic acid or 8-aminonaphthalene-1,3,6-trisulfonic acid, provided more accurate predictions of the retention data in comparison to native oligosaccharides or their neutral derivatives. The oligomeric mixed-mode model allowed prediction of gradient retention times using only one gradient profile, which significantly speeded-up the method development. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of the recently reported USP gradient HPLC method for analysis of anti-tuberculosis drugs for its ability to resolve degradation products of rifampicin.

PubMed

Mohan, Bhavika; Sharda, Nishi; Singh, Saranjit

2003-03-10

The recently notified USP gradient HPLC method for quantitative determination of rifampicin, isoniazid and pyrazinamide in fixed dose combination (FDC) formulations was evaluated to determine its ability to resolve major degradation products of rifampicin, viz. 3-formylrifamycin SV, rifampicin N-oxide, 25-desacetyl rifampicin, rifampicin quinone, and the newly reported isonicotinyl hydrazone, an interaction product of 3-formylrifamycin and isoniazid. The first observation was that the requirements of theoretical plates listed in the given method were met for rifampicin, but not for isoniazid and pyrazinamide, even on columns of different makes. The resolving power of the method was also dependent upon make of the column. On two of the three columns of the three tested, it was able to resolve most degradation products, except rifampicin N-oxide and 25-desacetylrifampicin, which were overlapping. The method was modified and an overall satisfactory resolution for all components was obtained by changing the buffer: organic modifier ratio of solution B in the gradient from 45:55 to 55:45 and decreasing the flow rate from 1.5 to 1.0 ml/min, keeping all other conditions constant.

Rapid separation and identification of anthocyanins from flowers of Viola yedoensis and V. prionantha by high-performance liquid chromatography-photodiode array detection-electrospray ionisation mass spectrometry.

PubMed

Zhang, Jie; Wang, Liang-Sheng; Gao, Jin-Ming; Xu, Yan-Jun; Li, Lian-Fang; Li, Chong-Hui

2012-01-01

Anthocyanins are important plant secondary metabolites. They show strong antioxidant activities and have potential as anti-cancer agents. Viola yedoensis and V. prionantha are traditional Chinese medicines and ornamental plants. However, the anthocyanin compositions of these two species are still unresolved. To develop a rapid and reliable high-performance liquid chromatography (HPLC) method for the separation and identification of anthocyanins from V. yedoensis and V. prionantha. Samples were extracted in methanol-water-formic acid-TFA (70:27:2:1, v/v). HPLC analysis was done on a C(18) column (TSK-GEL ODS-80Ts: 150 × 4.6 mm i.d.). Four solvent systems were tested to optimise the separation of anthocyanins using different gradient separation systems. HPLC-photodiode array detection (DAD) coupled to electrospray ionisation mass spectrometry (ESI-MS) was used to carry out the comprehensive characterisation of anthocyanins. Fourteen anthocyanins were characterised within 40 min with satisfactory peak resolution by a gradient composed of 10% aqueous formic acid and formic acid-acetonitrile-water (10:40:50, v/v). The calibration curve showed an excellent linear regression (r(2)  = 0.9995) and low intra- and inter-day variations (RSD < 3.67%). The detected anthocyanins derived from Dp, Cy, Pt, Mv and Pn, could be divided into three groups: non-acylated glycosides, acetylglycosides and coumaroylglycosides. Anthocyanins distribution exhibited remarkable differences in aglycone levels and acylation patterns. The optimised method was successfully applied for the analysis of 14 anthocyanins from V. yedoensis and V. prionantha. The identification of anthocyanin constitutions is valuable for breeding and will open up new prospects for their medicinal application. Copyright © 2011 John Wiley & Sons, Ltd.

Development and application of a validated HPLC method for the analysis of dissolution samples of levothyroxine sodium drug products.

PubMed

Collier, J W; Shah, R B; Bryant, A R; Habib, M J; Khan, M A; Faustino, P J

2011-02-20

A rapid, selective, and sensitive gradient HPLC method was developed for the analysis of dissolution samples of levothyroxine sodium tablets. Current USP methodology for levothyroxine (L-T(4)) was not adequate to resolve co-elutants from a variety of levothyroxine drug product formulations. The USP method for analyzing dissolution samples of the drug product has shown significant intra- and inter-day variability. The sources of method variability include chromatographic interferences introduced by the dissolution media and the formulation excipients. In the present work, chromatographic separation of levothyroxine was achieved on an Agilent 1100 Series HPLC with a Waters Nova-pak column (250 mm × 3.9 mm) using a 0.01 M phosphate buffer (pH 3.0)-methanol (55:45, v/v) in a gradient elution mobile phase at a flow rate of 1.0 mL/min and detection UV wavelength of 225 nm. The injection volume was 800 μL and the column temperature was maintained at 28°C. The method was validated according to USP Category I requirements. The validation characteristics included accuracy, precision, specificity, linearity, and analytical range. The standard curve was found to have a linear relationship (r(2)>0.99) over the analytical range of 0.08-0.8 μg/mL. Accuracy ranged from 90 to 110% for low quality control (QC) standards and 95 to 105% for medium and high QC standards. Precision was <2% at all QC levels. The method was found to be accurate, precise, selective, and linear for L-T(4) over the analytical range. The HPLC method was successfully applied to the analysis of dissolution samples of marketed levothyroxine sodium tablets. Published by Elsevier B.V.

Quantification of neutral human milk oligosaccharides by graphitic carbon HPLC with tandem mass spectrometry

PubMed Central

Bao, Yuanwu; Chen, Ceng; Newburg, David S.

2012-01-01

Defining the biologic roles of human milk oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral oligosaccharides of human milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon HPLC with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights allowing 11 peaks to be fully resolved and quantified by monitoring mass to charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 oligosaccharides is linear from 0.078 or 0.156 to 20 μg/mL (R2 > 0.998). Precision (CV) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, accurate quantification for each of the 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation. PMID:23068043

PREPARATIVE ISOLATION AND PURIFICATION OF CHEMICAL CONSTITUENTS OF BELAMCANDA BY MPLC, HSCCC AND PREP-HPLC

PubMed Central

Wang, Xiaohong; Liang, Yong; Peng, Cuilin; Xie, Huichun; Pan, Man; Zhang, Tianyou; Ito, Yoichiro

2010-01-01

Combined with medium-pressure liquid chromatography (MPLC) and preparative high-pressure liquid chromatography (Prep-HPLC), high-speed countercurrent chromatography (HSCCC) was successfully applied for separation and purification of isoflavonoids from the extract of belamcanda. HSCCC separation was performed on a two-phase solvent system composed of methyl tert-butyl ether -ethyl acetate - n-butyl alcohol – acetonitrile −0.1% aqueous trifluoroacetic acid at a volume radio of 1:2:1:1:5. Semi-purified peak fractions from HSCCC separation were further purified by Prep-HPLC. Nine well-separated fractions were analyzed by HPLC-UV absorption spectrometry to determine their purities and characterized with ESI-MSn. Except for peaksland VII (unknown) seven compounds were identified as apocynin (peak II), mangiferin (peak III), 7-O-methylmangiferin (peak IV), hispidulin (peak V), 3′-hydroxyltectoridin (peak VI), iristectorin B (peak VII), isoiridin (peak IX). PMID:21552369

Gradient separation of oligosaccharides and suppressing anomeric mutarotation with enhanced-fluidity liquid hydrophilic interaction chromatography.

PubMed

Bennett, Raffeal; Olesik, Susan V

2017-04-01

Enhanced fluidity liquid chromatography using the hydrophilic interaction retention mechanism (EFLC-HILIC) is studied as an alternative separation mode for analyzing oligosaccharides and other sugars. These carbohydrates, which are important for the study of foods and biological systems, are difficult to comprehensively profile and either require a non-green, expensive solvent (i.e. acetonitrile) or derivatization of the analytes at the expense of time, sample loss, and loss of quantitative information. These difficulties arise from the diverse isomerism, mutarotation, and lack of a useable chromophore/fluorophore for spectroscopic detection. Enhanced fluidity liquid chromatography is an alternative separation method that involves the use of conventional polar solvents, such as methanol/water mixtures, as the primary mobile phase component and liquid carbon dioxide (CO 2 ) as the modifier in subcritical conditions. The addition of liquid CO 2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis and higher efficiency. This work illustrates an optimized EFLC-HILIC separation of a test mixture of oligosaccharides and simple sugars with a resolution greater than 1.3 and an analysis time decrease of over 35% compared to a conventional HPLC HILIC-mode analysis using acetonitrile/water mobile phases. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of trace amounts of rare-earth elements in highly pure neodymium oxide by sector field inductively coupled plasma mass spectrometry (ICP-SFMS) and high-performance liquid chromatography (HPLC) techniques

NASA Astrophysics Data System (ADS)

Pedreira, W. R.; Sarkis, J. E. S.; da Silva Queiroz, C. A.; Rodrigues, C.; Tomiyoshi, I. A.; Abrão, A.

2003-02-01

Recently rare-earth elements (REE) have received much attention in fields of geochemistry and industry. Rapid and accurate determinations of them are increasingly required as industrial demands expand. Sector field inductively coupled plasma mass spectrometry (ICP-SFMS) with high-performance liquid chromatography (HPLC) has been applied to the determination of REE. HR ICP-MS was used as an element-selective detector for HPLC in highly pure materials. The separation of REE with HPLC helped to avoid erroneous analytical results due to spectral interferences. Sixteen elements (Sc, Y and 14 lanthanides) were determined selectively with the HPLC/ICP-SFMS system using a concentration gradient methods. The detection limits with the HPLC/ICP-SFMS system were about 0.5-10 pg mL-1. The percentage recovery ranged from 90% to 100% for different REE. The %RSD of the methods varying between 2.5% and 4.5% for a set of five (n=5) replicates was found for the IPEN's material and for the certificate reference sample. Determination of trace REEs in two highly pure neodymium oxides samples (IPEN and Johnson Matthey Company) were performed. In short, the IPEN's materials which are highly pure (>99.9%) were successfully analyzed without spectral interferences.

Separation and determination of minor photosynthetic pigments by reversed-phase HPLC with minimal alteration of chlorophylls.

PubMed

Nakamura, A; Watanabe, T

2001-04-01

Reversed-phase HPLC conditions for separation of chlorophyll (Chl) a, Chl a' (the C132-epimer of Chl a), pheophytin (Pheo) a (the primary electron acceptor of photosystem (PS) II), and phylloquinone (PhQ) (the secondary electron acceptor of PS 1), have been developed. Pigment extraction conditions were optimized in terms of pigment alteration and extraction efficiency. Pigment composition analysis of light-harvesting complex II, which would not contain Chl a' nor Pheo a, showed the Chl a'/Chl a ratio of 3-4 x 10(-4) and the Pheo a/Chl a ratio of 4-5 x 10(-4), showing that the conditions developed here were sufficiently inert for Chl analysis. Preliminary analysis of thylakoid membranes with this analytical system gave the PhQ/Chl a' ratio of 0.58 +/- 0.03 (n = 4), in line with the stoichiometry of one molecule of Chl a' per PS I.

HPLC determination of flavonoid glycosides in Mongolian Dianthus versicolor Fisch. (Caryophyllaceae) compared with quantification by UV spectrophotometry.

PubMed

Obmann, Astrid; Purevsuren, Sodnomtseren; Zehl, Martin; Kletter, Christa; Reznicek, Gottfried; Narantuya, Samdan; Glasl, Sabine

2012-01-01

Dianthus versicolor is used in traditional Mongolian medicine against liver impairment. Fractions enriched in flavone-di- and triglycosides were shown to enhance bile secretion. Therefore, reliable and accurate analytical methods are needed for the determination of these flavonoids in the crude drug and extracts thereof. To provide a validated HPLC-DAD (diode array detector) method especially developed for the separation of polar flavonoids and to compare the data obtained with those evaluated by UV spectrophotometry. Separations were carried out on an Aquasil® C₁₈-column (4.6 mm × 250.0 mm, 5 µm) with a linear gradient of acetonitrile and water (adjusted to pH 2.8 with trifluoroacetic acid) as mobile phase. Rutoside was employed as internal standard with linear behavior in a concentration range of 0.007-3.5 mg/mL. Accuracy was determined by spiking the crude drug with saponarin resulting in recoveries between 92% and 102%. The method allows the quantification of highly polar flavonoid glycosides and the determination of their total content. For saponarin a linear response was evaluated within the range 0.007-3.5 mg/mL (R²  > 0.9999). It was proven that threefold sonication represents a time-saving, effective and cheap method for the extraction of the polar flavonoid glycosides. The contents determined by HPLC were shown to be in agreement with those obtained employing UV spectrophotometry. The study has indicated that the newly developed HPLC method represents a powerful technique for the quality control of D. versicolor. Ultraviolet spectrophotometry may be used alternatively provided that the less polar flavonoids are removed by purification. Copyright © 2011 John Wiley & Sons, Ltd.

Generating and Separating Twisted Light by gradient-rotation Split-Ring Antenna Metasurfaces.

PubMed

Zeng, Jinwei; Li, Ling; Yang, Xiaodong; Gao, Jie

2016-05-11

Nanoscale compact optical vortex generators promise substantially significant prospects in modern optics and photonics, leading to many advances in sensing, imaging, quantum communication, and optical manipulation. However, conventional vortex generators often suffer from bulky size, low vortex mode purity in the converted beam, or limited operation bandwidth. Here, we design and demonstrate gradient-rotation split-ring antenna metasurfaces as unique spin-to-orbital angular momentum beam converters to simultaneously generate and separate pure optical vortices in a broad wavelength range. Our proposed design has the potential for realizing miniaturized on-chip OAM-multiplexers, as well as enabling new types of metasurface devices for the manipulation of complex structured light beams.

[Influences of ion-suppressors on retention behaviors of nine food additives in reversed-phase high performance liquid chromatographic separation].

PubMed

Zhao, Yonggang; Chen, Xiaohong; Li, Xiaoping; Yao, Shanshan; Jin, Micong

2011-10-01

The influences of ion-suppressors on retention behaviors of nine food additives, i.e., acesulfame, saccharin, caffeine, aspartame, benzoic acid, sorbic acid, stevioside, dehydroacetic acid and neotame in reversed-phase high performance liquid chromatographic (RP-HPLC) separation were investigated. The organic modification effects of acids, i. e. , trifluoroacetic acid (TFA) and buffer salts, i. e. , TFA-ammonium acetate (AmAc) were studied emphatically. The relationships between retention factors of solutes and volume percentages of ion-suppressors in the mobile phase systems of acetonitrile-TFA aqueous solution and acetonitrile-TFA-AmAc aqueous solution were quantitatively established, separately. The separation of nine food additives was completed by a gradient elution with acetonitrile-TFA (0.01%, v/v)-AmAc (2. 5 mmol/L) aqueous solution as the mobile phases. An RP-HPLC method was established for the simultaneous determination of nine food additives in red wine. In the range of 10. 0 - 100. 0 mg/L, nine food additives showed good linearity with the correlation coefficients ( r2 ) larger than 0. 999 1. The limits of detection (LODs) were in the range of 0. 33 - 2. 36 mg/L and the limits of quantification (LOQs) were in the range of 1. 11 - 7. 80 mg/L. The spiked recoveries were between 87. 61% and 108. 4% with the relative standard deviations (RSDs) of 2. 2% -9. 4%. These results are of referential significance for the rapid establishment and accu- rate optimization of RP-HPLC separation for the simultaneous determination of food additives in other foods.

HPLC retention thermodynamics of grape and wine tannins.

PubMed

Barak, Jennifer A; Kennedy, James A

2013-05-08

The effect of grape and wine tannin structure on retention thermodynamics under reversed-phase high-performance liquid chromatography conditions on a polystyrene divinylbenzene column was investigated. On the basis of retention response to temperature, an alternative retention factor was developed to approximate the combined temperature response of the complex, unresolvable tannin mixture. This alternative retention factor was based upon relative tannin peak areas separated by an abrupt change in solvent gradient. Using this alternative retention factor, retention thermodynamics were calculated. Van't Hoff relationships of the natural log of the alternative retention factor against temperature followed Kirchoff's relationship. An inverse quadratic equation was fit to the data, and from this the thermodynamic parameters for tannin retention were calculated. All tannin fractions exhibited exothermic, spontaneous interaction, with enthalpy-entropy compensation observed. Normalizing for tannin size, distinct tannin compositional effects on thermodynamic parameters were observed. The results of this study indicate that HPLC can be valuable for measuring the thermodynamics of tannin interaction with a hydrophobic surface and provides a potentially valuable alternative to calorimetry. Furthermore, the information gathered may provide insight into understanding red wine astringency quality.

Fast gradient separation by very high pressure liquid chromatography: reproducibility of analytical data and influence of delay between successive runs.

PubMed

Stankovicha, Joseph J; Gritti, Fabrice; Beaver, Lois Ann; Stevensona, Paul G; Guiochon, Georges

2013-11-29

Five methods were used to implement fast gradient separations: constant flow rate, constant column-wall temperature, constant inlet pressure at moderate and high pressures (controlled by a pressure controller),and programmed flow constant pressure. For programmed flow constant pressure, the flow rates and gradient compositions are controlled using input into the method instead of the pressure controller. Minor fluctuations in the inlet pressure do not affect the mobile phase flow rate in programmed flow. There producibilities of the retention times, the response factors, and the eluted band width of six successive separations of the same sample (9 components) were measured with different equilibration times between 0 and 15 min. The influence of the length of the equilibration time on these reproducibilities is discussed. The results show that the average column temperature may increase from one separation to the next and that this contributes to fluctuation of the results.

Novel surface modification of polymer-based separation media controlling separation selectivity, retentivity and generation of electroosmotic flow.

PubMed

Hosoya, Ken; Kubo, Takuya; Takahashi, Katsuo; Ikegami, Tohru; Tanaka, Nobuo

2002-12-06

Uniformly sized packing materials based on synthetic polymer particles for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) have been prepared from polymerization mixtures containing methacrylic acid (MAA) as a functional monomer and by using a novel surface modification method. This "dispersion method" affords effectively modified separation media. Both the amount of MAA utilized in the preparation and reaction time affect the selectivity of chromatographic separation in both the HPLC and the CEC mode and electroosmotic flow. This detailed study revealed that the dispersion method effectively modified internal surface of macroporous separation media and, based on the amount of MAA introduced, exclusion mechanism for the separation of certain solutes could be observed.

Development and validation of an RP-HPLC method for quantitative determination of vanillin and related phenolic compounds in Vanilla planifolia.

PubMed

Sinha, Arun Kumar; Verma, Subash Chandra; Sharma, Upendra Kumar

2007-01-01

A simple and fast method was developed using RP-HPLC for separation and quantitative determination of vanillin and related phenolic compounds in ethanolic extract of pods of Vanilla planifolia. Ten phenolic compounds, namely 4-hydroxybenzyl alcohol, vanillyl alcohol, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and piperonal were quantitatively determined using ACN, methanol, and 0.2% acetic acid in water as a mobile phase with a gradient elution mode. The method showed good linearity, high precision, and good recovery of compounds of interest. The present method would be useful for analytical research and for routine analysis of vanilla extracts for their quality control.

Retention prediction and separation optimization under multilinear gradient elution in liquid chromatography with Microsoft Excel macros.

PubMed

Fasoula, S; Zisi, Ch; Gika, H; Pappa-Louisi, A; Nikitas, P

2015-05-22

A package of Excel VBA macros have been developed for modeling multilinear gradient retention data obtained in single or double gradient elution mode by changing organic modifier(s) content and/or eluent pH. For this purpose, ten chromatographic models were used and four methods were adopted for their application. The methods were based on (a) the analytical expression of the retention time, provided that this expression is available, (b) the retention times estimated using the Nikitas-Pappa approach, (c) the stepwise approximation, and (d) a simple numerical approximation involving the trapezoid rule for integration of the fundamental equation for gradient elution. For all these methods, Excel VBA macros have been written and implemented using two different platforms; the fitting and the optimization platform. The fitting platform calculates not only the adjustable parameters of the chromatographic models, but also the significance of these parameters and furthermore predicts the analyte elution times. The optimization platform determines the gradient conditions that lead to the optimum separation of a mixture of analytes by using the Solver evolutionary mode, provided that proper constraints are set in order to obtain the optimum gradient profile in the minimum gradient time. The performance of the two platforms was tested using experimental and artificial data. It was found that using the proposed spreadsheets, fitting, prediction, and optimization can be performed easily and effectively under all conditions. Overall, the best performance is exhibited by the analytical and Nikitas-Pappa's methods, although the former cannot be used under all circumstances. Copyright © 2015 Elsevier B.V. All rights reserved.

Separation control in adverse pressure gradients using high-speed microjets

NASA Astrophysics Data System (ADS)

Kumar, Vikas

Inlets to aircraft propulsion systems must supply flow to the compressor with minimal pressure loss, flow distortion or unsteadiness. Flow separation in internal flows such as inlets and ducts in aircraft propulsion systems and external flows such as over aircraft wings, is undesirable as it reduces the overall system performance. The objective of present study is to understand the nature of separation and more importantly, to explore the applicability of high-speed microjets to actively control this flow separation. The geometry used for this experimental study was a generic backward facing "Stratford Ramp" equipped with arrays of high-speed microjets. The incoming flow was examined over a freestream velocity range of 10-65m/s and at ramp angle in range of 0-10°. It was observed that the flow separates at 30m/s and beyond for all angle of attack. The magnitude and extent of separation bubble increases with increasing adverse pressure gradients and/or increase in free-stream velocity. The separated flow for all the examined conditions was completely attached using suitable array of high-speed microjets. The most notable fact was that elimination of reverse velocity regions was accompanied by a reduction in flow unsteadiness and increased two-dimensionality in the flow. In particular, these gains were achieved with a minimal mass flux, less than 0.2% of the primary flow based on 30% Boundary Layer Ingesting duct. Detailed measurements were obtained to understand the flow control dynamics. The control effectiveness was found to be dependent on the actuation location with respect to separation, jet to cross-flow momentum ratio and the angle at which microjets supply the momentum. It was also determined that the control effect of the microjets, in part, is due to creation of strong stream-wise vortices which enhance the mixing between low-momentum fluid closer to the surface and high-momentum fluid further away from the surface. The penetration depth of microjets was

HPLC-ESI-QTOF-MS as a powerful analytical tool for characterising phenolic compounds in olive-leaf extracts.

PubMed

Quirantes-Piné, Rosa; Lozano-Sánchez, Jesús; Herrero, Miguel; Ibáñez, Elena; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

2013-01-01

Olea europaea L. leaves may be considered a cheap, easily available natural source of phenolic compounds. In a previous study we evaluated the possibility of obtaining bioactive phenolic compounds from olive leaves by pressurised liquid extraction (PLE) for their use as natural anti-oxidants. The alimentary use of these kinds of extract makes comprehensive knowledge of their composition essential. To undertake a comprehensive characterisation of two olive-leaf extracts obtained by PLE using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS). Olive leaves were extracted by PLE using ethanol and water as extraction solvents at 150°C and 200°C respectively. Separation was carried out in a HPLC system equipped with a C₁₈-column working in a gradient elution programme coupled to ESI-QTOF-MS operating in negative ion mode. This analytical platform was able to detect 48 compounds and tentatively identify 31 different phenolic compounds in these extracts, including secoiridoids, simple phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C was also identified for the first time in olive leaves. The coupling of HPLC-ESI-QTOF-MS led to the in-depth characterisation of the olive-leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical tool is very valuable in the study of phenolic compounds in plant matrices. Copyright © 2012 John Wiley & Sons, Ltd.

[A new HPLC procedure for cyclamate in food with pre-chromatographic derivatization].

PubMed

Schwedt, G; Hauck, M

1988-08-01

A high-pressure liquid chromatography (HPLC) procedure for the detection of cyclamate in liquid and solid samples is presented, which depends on oxidation and the reaction of cyclohexylamine with o-phthaldialdehyde to form a condensation product. The results of the HPLC analysis, using an RP-C 18 separation system with UV detection at 242 nm are reported. Contents, from 2 to 400 mg/l, can be detected in less than 2 h (HPLC analysis within 20 min) with relative standard deviations of 4%. Only for cucumber infusions were incomplete recoveries of 68% obtained.

Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast.

PubMed

Méndez, S P; González, E B; Sanz-Medel, A

2001-05-01

Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast. Copyright 2001 John Wiley & Sons, Ltd.

Quantitative Clinical Diagnostic Analysis of Acetone in Human Blood by HPLC: A Metabolomic Search for Acetone as Indicator

PubMed Central

Akgul Kalkan, Esin; Sahiner, Mehtap; Ulker Cakir, Dilek; Alpaslan, Duygu; Yilmaz, Selehattin

2016-01-01

Using high-performance liquid chromatography (HPLC) and 2,4-dinitrophenylhydrazine (2,4-DNPH) as a derivatizing reagent, an analytical method was developed for the quantitative determination of acetone in human blood. The determination was carried out at 365 nm using an ultraviolet-visible (UV-Vis) diode array detector (DAD). For acetone as its 2,4-dinitrophenylhydrazone derivative, a good separation was achieved with a ThermoAcclaim C18 column (15 cm × 4.6 mm × 3 μm) at retention time (t R) 12.10 min and flowrate of 1 mL min−1 using a (methanol/acetonitrile) water elution gradient. The methodology is simple, rapid, sensitive, and of low cost, exhibits good reproducibility, and allows the analysis of acetone in biological fluids. A calibration curve was obtained for acetone using its standard solutions in acetonitrile. Quantitative analysis of acetone in human blood was successfully carried out using this calibration graph. The applied method was validated in parameters of linearity, limit of detection and quantification, accuracy, and precision. We also present acetone as a useful tool for the HPLC-based metabolomic investigation of endogenous metabolism and quantitative clinical diagnostic analysis. PMID:27298750

Development and validation of an HPLC method for tetracycline-related USP monographs.

PubMed

Hussien, Emad M

2014-09-01

A novel reversed-phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4-epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4-epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H3 PO4 as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear over the range 80-120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50-150% of the acceptance criteria specified in the individual USP monographs for 4-epianhydrotetracycline hydrochloride. The limit of quantification for 4-epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust. Copyright © 2014 John Wiley & Sons, Ltd.

PLETHORA gradient formation mechanism separates auxin responses.

PubMed

Mähönen, Ari Pekka; Ten Tusscher, Kirsten; Siligato, Riccardo; Smetana, Ondřej; Díaz-Triviño, Sara; Salojärvi, Jarkko; Wachsman, Guy; Prasad, Kalika; Heidstra, Renze; Scheres, Ben

2014-11-06

During plant growth, dividing cells in meristems must coordinate transitions from division to expansion and differentiation, thus generating three distinct developmental zones: the meristem, elongation zone and differentiation zone. Simultaneously, plants display tropisms, rapid adjustments of their direction of growth to adapt to environmental conditions. It is unclear how stable zonation is maintained during transient adjustments in growth direction. In Arabidopsis roots, many aspects of zonation are controlled by the phytohormone auxin and auxin-induced PLETHORA (PLT) transcription factors, both of which display a graded distribution with a maximum near the root tip. In addition, auxin is also pivotal for tropic responses. Here, using an iterative experimental and computational approach, we show how an interplay between auxin and PLTs controls zonation and gravitropism. We find that the PLT gradient is not a direct, proportionate readout of the auxin gradient. Rather, prolonged high auxin levels generate a narrow PLT transcription domain from which a gradient of PLT protein is subsequently generated through slow growth dilution and cell-to-cell movement. The resulting PLT levels define the location of developmental zones. In addition to slowly promoting PLT transcription, auxin also rapidly influences division, expansion and differentiation rates. We demonstrate how this specific regulatory design in which auxin cooperates with PLTs through different mechanisms and on different timescales enables both the fast tropic environmental responses and stable zonation dynamics necessary for coordinated cell differentiation.

Quantitative separation of the anisotropic magnetothermopower and planar Nernst effect by the rotation of an in-plane thermal gradient

NASA Astrophysics Data System (ADS)

Reimer, Oliver; Meier, Daniel; Bovender, Michel; Helmich, Lars; Dreessen, Jan-Oliver; Krieft, Jan; Shestakov, Anatoly S.; Back, Christian H.; Schmalhorst, Jan-Michael; Hütten, Andreas; Reiss, Günter; Kuschel, Timo

2017-01-01

A thermal gradient as the driving force for spin currents plays a key role in spin caloritronics. In this field the spin Seebeck effect (SSE) is of major interest and was investigated in terms of in-plane thermal gradients inducing perpendicular spin currents (transverse SSE) and out-of-plane thermal gradients generating parallel spin currents (longitudinal SSE). Up to now all spincaloric experiments employ a spatially fixed thermal gradient. Thus, anisotropic measurements with respect to well defined crystallographic directions were not possible. Here we introduce a new experiment that allows not only the in-plane rotation of the external magnetic field, but also the rotation of an in-plane thermal gradient controlled by optical temperature detection. As a consequence, the anisotropic magnetothermopower and the planar Nernst effect in a permalloy thin film can be measured simultaneously. Thus, the angular dependence of the magnetothermopower with respect to the magnetization direction reveals a phase shift, that allows the quantitative separation of the thermopower, the anisotropic magnetothermopower and the planar Nernst effect.

Simultaneous separation and analysis of water- and fat-soluble vitamins on multi-modal reversed-phase weak anion exchange material by HPLC-UV.

PubMed

Dabre, Romain; Azad, Nazanin; Schwämmle, Achim; Lämmerhofer, Michael; Lindner, Wolfgang

2011-04-01

Several methods for the separation of vitamins on HPLC columns were already validated in the last 20 years. However, most of the techniques focus on separating either fat- or water-soluble vitamins and only few methods are intended to separate lipophilic and hydrophilic vitamins simultaneously. A mixed-mode reversed-phase weak anion exchange (RP-WAX) stationary phase was developed in our laboratory in order to address such mixture of analytes with different chemical characteristics, which are difficult to separate on standard columns. The high versatility in usage of the RP-WAX chromatographic material allowed a baseline separation of ten vitamins within a single run, seven water-soluble and three fat-soluble, using three different chromatographic modes: some positively charged vitamins are eluted in ion exclusion and ion repulsion modes whereas the negatively charged molecules are eluted in the ion exchange mechanism. The non-charged molecules are eluted in a classical reversed-phase mode, regarding their polarities. The method was validated for the vitamin analysis in tablets, evaluating selectivity, robustness, linearity, accuracy, and precision. The validated method was finally employed for the analysis of the vitamin content of some commercially available supplement tablets. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2-deoxyglucose in human plasma

PubMed Central

Gounder, Murugesan K.; Lin, Hongxia; Stein, Mark; Goodin, Susan; Bertino, Joseph R.; Kong, Ah-Ng Tony; DiPaola, Robert S.

2015-01-01

2-deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with the glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate/boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45min. The analytes were separated on a YMC ODS C18 reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425nm. The 2-DG calibration curves were linear over the range of 0.63 to 300μg/mL with the limit of detection of 0.5μg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8% and the accuracy ranged from 86.8% to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors. PMID:21932382

A nitromethane-based HPLC system alternative to acetonitrile for carotenoid analysis of fruit and vegetables.

PubMed

Sandmann, Gerhard

2010-01-01

Acetonitrile-based HPLC systems are the most commonly used for carotenoid analysis from different plant tissues. Because of the acetonitrile shortage, an HPLC system for the separation of carotenoids on C(18) reversed-phase columns was developed in which an acetonitrile-alcohol-based mobile phase was replaced by nitromethane. This solvent comes closest to acetonitrile with respect to its elutrophic property. Our criterion was to obtain similar separation and retention times for a range of differently structured carotenoids. This was achieved by further increase in the lipophilicity with ethylacetate. For all the carotenoids which we tested, we found co-elution only of β-cryptoxanthin and lycopene. By addition of 1% of water, separation of this pair of carotenoids was also achieved. The final recommended mobile phase consisted of nitromethane : 2-propanol : ethyl acetate : water (79 : 10 : 10 : 1, by volume). On Nucleosil C(18) columns and related ones like Hypersil C(18), we obtained separation of carotenes, hydroxyl, epoxy and keto derivatives, which resembles the excellent separation properties of acetonitrile-based mobile phases on C(18) reversed phase columns. We successfully applied the newly developed HPLC system to the separation of carotenoids from different vegetables and fruit. Copyright © 2010 John Wiley & Sons, Ltd.

Identification and Characterization of Asulam Impurities in Self Made Bulk Batch Synthesis and Quantification by RP-HPLC Method.

PubMed

Mahaboob Basha, D; Venkata Reddy, G; Gopi Krishna, Y; Kumara Swamy, B E; Vijay, Rajani

2018-04-19

The first approach of this research paper explores the simultaneous characterization and determination of theAsulam active ingredient and its associated nine impurities in bulk batch production by the gradient reverse-phase high-performance liquid chromatographic (RP-HPLC) method. The best separation from its potential impurities and reproducible method was achieved by selecting the Cosmosil C-18 (250 × 4.6 mm, 5 μm particle size) analytical column with a run time of 40 min. The pumping chromatographic mobile phase was composed of 0.1% formic acid in milli-Q water (pH ~2.72) and methanol (80 + 20, v/v). An ambient column-oven temperature and UV detection at 260 nm were used. For this broad resolution, a gradient program was employed at a flow rate of 1.20 mL/min. All potential related substances in Asulam bulk manufacturing were ascertained by mass, proton nuclear magnetic resonance, and infrared spectroscopy. The developed HPLC method was validated with respect to linearity (25.64-151.83 mg/L for Asulam and 0.71-16.29, 1.02-12.26, 1.01-20.29, 0.60-10.01, 1.04-16.65, 0.94-22.47, 0.93-16.60, 1.00-12.45, 1.00-12.45, and 0.71-12.17 mg/L for Impurities A to I with a correlation coefficient 0.999 for Asulam and all the impurities), precision (RSD, % for active analyte Asulam and impurities were ˂2%), accuracy (percent recovery for Asulam at two levels ranged from 99.28 to 99.35%, and for Impurities A to I, it was 93.44 to 101.41%), and specificity. Hence, this simple and reliable HPLC method was able to determine the purity of Asulam active analyte and the level of impurities in bulk batch synthesis. By using this quantified procedure, five self-made production batches were analyzed simultaneously.

High-throughput separation of cells by dielectrophoresis enhanced with 3D gradient AC electric field.

PubMed

Tada, Shigeru; Hayashi, Masako; Eguchi, Masanori; Tsukamoto, Akira

2017-11-01

We propose a novel, high-performance dielectrophoretic (DEP) cell-separation flow chamber with a parallel-plate channel geometry. The flow chamber, consisting of a planar electrode on the top and an interdigitated-pair electrode array at the bottom, was developed to facilitate the separation of cells by creating a nonuniform AC electric field throughout the volume of the flow chamber. The operation and performance of the device were evaluated using live and dead human epithermal breast (MCF10A) cells. The separation dynamics of the cell suspension in the flow chamber was also investigated by numerically simulating the trajectories of individual cells. A theoretical model to describe the dynamic cell behavior under the action of DEP, including dipole-dipole interparticle, viscous, and gravitational forces, was developed. The results demonstrated that the live cells traveling through the flow chamber congregated into sites where the electric field gradient was minimal, in the middle of the flow stream slightly above the centerlines of the grounded electrodes at the bottom. Meanwhile, the dead cells were trapped on the edges of the high-voltage electrodes at the bottom. Cells were thus successfully separated with a remarkably high separation ratio (∼98%) at the appropriately tuned field frequency and applied voltage. The numerically predicted behavior and spatial distribution of the cells during separation also showed good agreement with those observed experimentally.

Chiral separation and chemical profile of Dengzhan Shengmai by integrating comprehensive with multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

PubMed

Sheng, Ning; Zheng, Hao; Xiao, Yao; Wang, Zhe; Li, Menglin; Zhang, Jinlan

2017-09-29

Chemical profile for Chinese medicine formulas composed of several herbs is always a challenge due to a big array of small molecules with high chemical diversity so much as isomers. The present paper develops a feasible strategy to characterize and identify complex chemical constituents of a four-herb traditional Chinese medicine formula, Denzhan Shenmai (DZSM) by integrating comprehensive two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC×LC-qTOF-MS) with multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (MHC-qTOF-MS). DZSM was separated by C8×C18 HPLC column system for comprehensive two-dimensional liquid chromatography system and 283 compounds most of which belonged to phenolic acid, flavonoid, saponin and lignan families were characterized and identified within 75min. Some isomers and compounds at low level were analyzed on C8×Chiral HPLC column system for multiple heart-cutting two-dimensional liquid chromatography system with 1D and 2D optimized gradient elution program. These 1D cutting fractions were successively separated on 2D chiral chromatographic column under extended the 2D gradient elution time from 30s to 5.0min. 12 pairs of isomer compounds were separated with good resolution. The combination of LC×LC and MHC system provides a powerful technique for global chemical profiling of DZSM and provided feasible strategy for other complex systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Separation of anionic oligosaccharides by high-performance liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Green, E.D.; Baenziger, J.U.

1986-10-01

The authors have developed methods for rapid fractionation of anionic oligosaccharides containing sulfate and/or sialic acid moieties by high-performance liquid chromatography (HPLC). Ion-exchange HPLC on amine-bearing columns (Micropak AX-10 and AX-5) at pH 4.0 is utilized to separate anionic oligosaccharides bearing zero, one, two, three, or four charges, independent of the identity of the anionic moieties (sulfate and/or sialic acid). Ion-exchange HPLC at pH 1.7 allows separation of neutral, mono-, di-, and tetrasialylated, monosulfated, and disulfated oligosaccharides. Oligosaccharides containing three sialic acid residues and those bearing one each of sulfate and sialic acid, however, coelute at pH 1.7. Since themore » latter two oligosaccharide species separate at pH 4.0, analysis at pH 4.0 followed by analysis at pH 1.7 can be utilized to completely fractionate complex mixtures of sulfated and sialylated oligosaccharides. Ion-suppression amine adsorption HPLC has previously been shown to separate anionic oligosaccharides on the basis of net carbohydrate content (size). In this study they demonstrate the utility of ion-suppression amine adsorption HPLC for resolving sialylated oligosaccharide isomers which differ only in the linkages of sialic acid residues (..cap alpha..2,3 vs ..cap alpha..2,6) and/or location of ..cap alpha..2,3- and ..cap alpha..2,6-linked sialic acid moieties on the peripheral branches of oligosaccharides. These two methods can be used in tandem to separate oligosaccharides, both analytically and preparatively, based on their number, types, and linkages of anionic moieties.« less

“Measure Your Gradient”: A New Way to Measure Gradients in High Performance Liquid Chromatography by Mass Spectrometric or Absorbance Detection

PubMed Central

Magee, Megan H.; Manulik, Joseph C.; Barnes, Brian B.; Abate-Pella, Daniel; Hewitt, Joshua T.; Boswell, Paul G.

2014-01-01

The gradient produced by an HPLC is never the same as the one it is programmed to produce, but non-idealities in the gradient can be taken into account if they are measured. Such measurements are routine, yet only one general approach has been described to make them: both HPLC solvents are replaced with water, solvent B is spiked with 0.1% acetone, and the gradient is measured by UV absorbance. Despite the widespread use of this procedure, we found a number of problems and complications with it, mostly stemming from the fact that it measures the gradient under abnormal conditions (e.g. both solvents are water). It is also generally not amenable to MS detection, leaving those with only an MS detector no way to accurately measure their gradients. We describe a new approach called “Measure Your Gradient” that potentially solves these problems. One runs a test mixture containing 20 standards on a standard stationary phase and enters their gradient retention times into open-source software available at www.measureyourgradient.org. The software uses the retention times to back-calculate the gradient that was truly produced by the HPLC. Here we present a preliminary investigation of the new approach. We found that gradients measured this way are comparable to those measured by a more accurate, albeit impractical, version of the conventional approach. The new procedure worked with different gradients, flow rates, column lengths, inner diameters, on two different HPLCs, and with six different batches of the standard stationary phase. PMID:25441073

Novel separation method for highly sensitive speciation of cancerostatic platinum compounds by HPLC-ICP-MS.

PubMed

Hann, S; Stefánka, Zs; Lenz, K; Stingeder, G

2005-01-01

A high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method is presented for analysis of cisplatin, monoaquacisplatin, diaquacisplatin, carboplatin, and oxaliplatin in biological and environmental samples. Chromatographic separation was achieved on pentafluorophenylpropyl-functionalized silica gel. For cisplatin, carboplatin, and oxaliplatin limits of detection of 0.09, 0.10, and 0.15 microg L(-1), respectively, were calculated at m/z 194, using aqueous standard solutions. (3 microL injection volume). The method was utilized for model experiments studying the stability of carboplatin and oxaliplatin at different chloride concentrations simulating wastewater and surface water conditions. It was found that a high fraction of carboplatin is stable in ultrapure water and in solutions containing 1.5 mol L(-1) Cl-, whereas oxaliplatin degradation was increased by increasing the chloride concentration. In order to support the assessment of oxaliplatin eco-toxicology, the method was tested for speciation of patient urine. The urine sample contained more than 17 different reaction products, which demonstrates the extensive biotransformation of the compound. In a second step of the study the method was successfully evaluated for monitoring cancerostatic platinum compounds in hospital waste water.

The use of dihexyldithiocarbamate in reverse-phase HPLC of metal chelates

NASA Astrophysics Data System (ADS)

Fatimah, S. S.; Bahti, H. H.; Hastiawan, I.; Permanasari, A.

2018-05-01

Dialkyldithiocarbamates have long been used as chelating agents in reverse-phase HPLC of transition metals. In the previous study, an alkyl homolog of this type of ligand, namely dihexyldithiocarbamate (DHDTC), was synthesized and characterized. The use of this particular ligand in the revese-phase HPLC of some selected transition metal ions is now reported for the first time. The mobile phase comprising of the flow rate and of the detection, in the separation of the metal chelates of Cd (II), Fe (III), Cu (II), and Co (III), were investigated on a C-18 column. The results showed that dihexylditiocarbamate could be used for separating Cd (II), Fe(III), Cu(II), and Co(III). Therefore, it could be used in simultaneous analysis.

Separation and characterization of polycyclic aromatic hydrocarbons and alkylphenols in coal derived solvents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hurtubise, R.J.; Allen, T.W.; Hussain, A.

1981-03-29

Dry-column chromatography with an aluminum oxide stationary phase and a n-hexane-ether (19:1) mobile phase was used to separate polycyclic aromatic hydrocarbons (PAH) by ring size. Prior to the dry-column chromatography step, the coal derived solvents were added to an acid treated silica gel column and eluted with chloroform. This step removed pyridine-type nitrogen heterocycles. After separation of the individual ring fractions, the fractions were further separated by either thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). If TLC was used, then after separation fluorescence profiles of each PAH ring fraction distributed on 30%-acetylated cellulose chromatoplates were obtained withmore » a spectrodensitometer. Measurement of fluorescence peak heights gave an approximate measure of the amount of the 3-, 4-, 5-, and 6- ring PAH. For HPLC separation, the 3- and 4- ring PAH fractions obtained from the dry-column chromatography step were separated with a ..mu..-Bondapak C/sub 18/ column and methanol:water (65:35) mobile phase. The HPLC separated PAH were characterized by chromatographic correlation factors and corrected fluorescence excitation spectra. Alkylphenols were identified in coal recycle solvent sample following separation by HPLC.« less

Intrinsic advantages of packed capillaries over narrow-bore columns in very high-pressure gradient liquid chromatography.

PubMed

Gritti, Fabrice; McDonald, Thomas; Gilar, Martin

2016-06-17

250μm×100mm fused silica glass capillaries were packed with 1.8μm high-strength silica (HSS) fully porous particles. They were prepared without bulky stainless steel endfittings and metal frits, which both generate significant sample dispersion. The isocratic efficiencies and gradient peak capacities of these prototype capillary columns were measured for small molecules (n-alkanophenones) using a home-made ultra-low dispersive micro-HPLC instrument. Their resolution power was compared to that of standard 2.1mm×100mm very high-pressure liquid chromatography (vHPLC) narrow-bore columns packed with the same particles. The results show that, for the same column efficiency (25000 plates) and gradient steepness (0.04min(-1)), the peak capacity of the 250μm i.d. capillary columns is systematically 15-20% higher than that of the 2.1mm i.d. narrow-bore columns. A validated model of gradient chromatography enabled one to predict accurately the observed peak capacities of the capillary columns for non-linear solvation strength retention behavior and under isothermal conditions. Thermodynamics applied to the eluent quantified the temperature difference for the thermal gradients in both capillary and narrow-bore columns. Experimental data revealed that the gradient peak capacity is more affected by viscous heating than the column efficiency. Unlike across 2.1mm i.d. columns, the changes in eluent composition across the 250μm i.d. columns during the gradient is rapidly relaxed by transverse dispersion. The combination of (1) the absence of viscous heating and (2) the high uniformity of the eluent composition across the diameter of capillary columns explains the intrinsic advantage of capillary over narrow-bore columns in gradient vHPLC. Copyright © 2016 Elsevier B.V. All rights reserved.

Short communication: separation and quantification of caseins and casein macropeptide using ion-exchange chromatography.

PubMed

Holland, B; Rahimi Yazdi, S; Ion Titapiccolo, G; Corredig, M

2010-03-01

The aim of this work was to improve an existing method to separate and quantify the 4 major caseins from milk samples (i.e., containing whey proteins) using ion-exchange chromatography. The separation process was carried out using a mini-preparative cation exchange column (1 or 5mL of column volume), using urea acetate as elution buffer at pH 3.5 with a NaCl gradient. All 4 major caseins were separated, and the purity of each peak was assessed using sodium dodecyl sulfate-PAGE. Purified casein fractions were also added to raw milk to confirm their elution volumes. The quantification was carried out using purified caseins in buffer as well as added directly to fresh skim milk. This method can also be employed to determine the decrease in kappa-casein and the release of the casein-macropeptide during enzymatic hydrolysis using rennet. In this case, the main advantage of using this method is the lack of organic solvents compared with the conventional method for separation of macropeptide (using reversed phase HPLC).

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue

NASA Technical Reports Server (NTRS)

Slocum, R. D.; Flores, H. E.; Galston, A. W.; Weinstein, L. H.

1989-01-01

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

The Effect of Column and Eluent Fluorination on the Retention and Separation of non-Fluorinated Amino Acids and Proteins by HPLC

PubMed Central

Joyner, Katherine; Wang, Weizhen; Yu, Yihua Bruce

2011-01-01

The effect of column and eluent fluorination on the retention and separation of non-fluorinated amino acids and proteins in HPLC is investigated. A side-by-side comparison of fluorocarbon column and eluents (F-column and F-eluents) with their hydrocarbon counterparts (H-column and H-eluents) in the separation of a group of 33 analytes, including 30 amino acids and 3 proteins, is conducted. The H-column and the F-column contain the n-C8H17 group and n-C8F17 group, respectively, in their stationary phases. The H-eluents include ethanol (EtOH) and isopropanol (ISP) while the F-eluents include trifluoroethanol (TFE) and hexafluorosopropanol (HFIP). The 2 columns and 4 eluents generated 8 (column, eluent) pairs that produce 264 retention time data points for the 33 analytes. A statistical analysis of the retention time data reveals that although the H-column is better than the F-column in analyte separation and H-eluents are better than F-eluents in analyte retention, the more critical factor is the proper pairing of column with eluent. Among the conditions explored in this project, optimal retention and separation is achieved when the fluorocarbon column is paired with ethanol, even though TFE is the most polar one among the 4 eluents. This result shows fluorocarbon columns have much potential in chromatographic analysis and separation of non-fluorinated amino acids and proteins. PMID:21318121

Arsenic-containing fatty acids and hydrocarbons in marine oils - determination using reversed-phase HPLC-ICP-MS and HPLC-qTOF-MS.

PubMed

Sele, Veronika; Sloth, Jens J; Holmelid, Bjarte; Valdersnes, Stig; Skov, Kasper; Amlund, Heidi

2014-04-01

Arsenolipids are the major arsenic species present in marine oils. Several structures of arsenolipids have been elucidated the last 5 years, demonstrating the chemical complexity of this trace element in the marine environment. Several commercial fish oils and marine oils, ranging in total arsenic concentrations from 1.6 to 12.5 mg kg(-1) oil, were analyzed for arsenolipids using reversed-phase high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The arsenolipids were quantified using three different arsenic-containing calibration standards; dimethylarsinate (DMA), triphenylarsinoxide (Ph₃AsO) and a synthesized arsenic-containing hydrocarbon (AsHC) (dimethylarsinoyl nonadecane; C₂₁H₄₃AsO). The observed variation in signal intensity for arsenic during the gradient elution profile in reversed-phase HPLC was compensated for by determining the time-resolved response factors for the arsenolipids. Isotopes of germanium ((74)Ge) and indium ((115)In) were suited as internal standards for arsenic, and were used for verification of the arsenic signal response factors during the gradient elution. Dimethylarsinate was the most suitable calibration standard for the quantification of arsenolipids, with recoveries between 91% and 104% compared to total arsenic measurements in the same extracts. A range of marine oils was investigated, including oils of several fish species, cod liver and seal, as well as three commercial fish oils. The AsHCs - C₁₇H₃₈AsO, C₁₉H₄₂AsO and C₂₃H₃₈AsO - were identified as the major arsenolipids in the extracts of all oils by HPLC coupled with quadrupole time-of-flight mass spectrometry (qTOF-MS). Minor amounts of two arsenic-containing fatty acids (AsFAs) (C₂₃H₃₈AsO₃ and C₂₄H₃₈AsO₃) were also detected in the oils. The sum of the AsHCs and the AsFAs determined in the present study accounted for 17-42% of the total arsenic in the oils

Application of quality by design concept to develop a dual gradient elution stability-indicating method for cloxacillin forced degradation studies using combined mixture-process variable models.

PubMed

Zhang, Xia; Hu, Changqin

2017-09-08

Penicillins are typical of complex ionic samples which likely contain large number of degradation-related impurities (DRIs) with different polarities and charge properties. It is often a challenge to develop selective and robust high performance liquid chromatography (HPLC) methods for the efficient separation of all DRIs. In this study, an analytical quality by design (AQbD) approach was proposed for stability-indicating method development of cloxacillin. The structures, retention and UV characteristics rules of penicillins and their impurities were summarized and served as useful prior knowledge. Through quality risk assessment and screen design, 3 critical process parameters (CPPs) were defined, including 2 mixture variables (MVs) and 1 process variable (PV). A combined mixture-process variable (MPV) design was conducted to evaluate the 3 CPPs simultaneously and a response surface methodology (RSM) was used to achieve the optimal experiment parameters. A dual gradient elution was performed to change buffer pH, mobile-phase type and strength simultaneously. The design spaces (DSs) was evaluated using Monte Carlo simulation to give their possibility of meeting the specifications of CQAs. A Plackett-Burman design was performed to test the robustness around the working points and to decide the normal operating ranges (NORs). Finally, validation was performed following International Conference on Harmonisation (ICH) guidelines. To our knowledge, this is the first study of using MPV design and dual gradient elution to develop HPLC methods and improve separations for complex ionic samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous Quantification of Limonin, Two Indolequinazoline Alkaloids, and Four Quinolone Alkaloids in Evodia rutaecarpa (Juss.) Benth by HPLC-DAD Method.

PubMed

Zhang, Pei-Ting; Pan, Bi-Yan; Liao, Qiong-Feng; Yao, Mei-Cun; Xu, Xin-Jun; Wan, Jin-Zhi; Liu, Dan; Xie, Zhi-Yong

2013-01-01

A simple and efficient HPLC-DAD (225 nm) method was developed and validated for the simultaneous determination of limonin and six key alkaloids (evodiamine, rutaecarpine, 1-methyl-2-undecyl-4(1H)-quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine) in Evodia rutaecarpa (Juss.) Benth, which has been widely used as one of the Traditional Chinese Medicines. The chromatographic separation was carried out on a Hypersil BDS C18 column, and gradient elution was employed with a mobile phase containing acetonitrile and water. Contents of the analytes in 18 batches of samples were analyzed by ultrasonic extraction with ethanol and water mixture (80 : 20, v/v) followed by HPLC analysis. Separation of the seven analytes was achieved within 60 min with good linearity (r > 0.999). The RSD of both the intraday and interday precision was below 1.85%. The accuracy at different concentrations was within the range of 97.91 to 100.49%. Hierarchical clustering analysis was performed to differentiate and classify the samples based on the contents of the seven constituents. This study indicated that the quality control of E. rutaecarpa could be simplified to the measurement of four constituents, and that limonin, 1-methyl-2-undecyl-4(1H)-quinolone, and dihydroevocarpine should also be served as the chemical markers together with evodiamine for the quality control of Evodia rutaecarpa (Juss.) Benth.

Simultaneous Quantification of Limonin, Two Indolequinazoline Alkaloids, and Four Quinolone Alkaloids in Evodia rutaecarpa (Juss.) Benth by HPLC-DAD Method

PubMed Central

Zhang, Pei-ting; Pan, Bi-yan; Liao, Qiong-feng; Yao, Mei-cun; Xu, Xin-jun; Wan, Jin-zhi; Liu, Dan; Xie, Zhi-yong

2013-01-01

A simple and efficient HPLC-DAD (225 nm) method was developed and validated for the simultaneous determination of limonin and six key alkaloids (evodiamine, rutaecarpine, 1-methyl-2-undecyl-4(1H)-quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine) in Evodia rutaecarpa (Juss.) Benth, which has been widely used as one of the Traditional Chinese Medicines. The chromatographic separation was carried out on a Hypersil BDS C18 column, and gradient elution was employed with a mobile phase containing acetonitrile and water. Contents of the analytes in 18 batches of samples were analyzed by ultrasonic extraction with ethanol and water mixture (80 : 20, v/v) followed by HPLC analysis. Separation of the seven analytes was achieved within 60 min with good linearity (r > 0.999). The RSD of both the intraday and interday precision was below 1.85%. The accuracy at different concentrations was within the range of 97.91 to 100.49%. Hierarchical clustering analysis was performed to differentiate and classify the samples based on the contents of the seven constituents. This study indicated that the quality control of E. rutaecarpa could be simplified to the measurement of four constituents, and that limonin, 1-methyl-2-undecyl-4(1H)-quinolone, and dihydroevocarpine should also be served as the chemical markers together with evodiamine for the quality control of Evodia rutaecarpa (Juss.) Benth. PMID:23738236

Direct-injection HPLC method of measuring micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column.

PubMed

Uranishi, Hiroaki; Nakamura, Mitsuhiro; Nakamura, Hiroki; Ikeda, Yukari; Otsuka, Mayuko; Kato, Zenichiro; Tsuchiya, Teruo

2011-04-15

A direct-injection HPLC-based method has been developed for determining amounts of micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column. The method is easy to perform and requires only 10 μL of a filtered plasma sample. The chromatographic separations were carried out with a gradient mode. The fluorescence detection wavelengths of excitation and emission were set at 273 nm and 464 nm, respectively. Retention times for micafungin and IS were 22.4 and 23.7 min, respectively. Micafungin and FR195743 (IS) peaks were completely separated with little tailing, and no interference was observed. The calibration curve of micafungin showed good linearity in the range of 0.5-20.0 μg/mL (r(2)=1.00). The intra-day accuracy ranged from -4.5 to 5.3%. The inter-day accuracy ranged from -9.8 to 1.5%. The precisions were less than 10%. This method is useful for the determination of micafungin in human plasma. Copyright © 2011 Elsevier B.V. All rights reserved.

Methods and applications of HPLC-AMS (WBio 5)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bucholz, B A; Clifford, A J; Duecker, S R

Pharmacokinetics of physiologic doses of nutrients, pesticides, and herbicides can easily be traced in humans using a {sup 14}C-labelled compound. Basic kinetics can be monitored in blood or urine by measuring the elevation in the {sup 14}C content above the control predose tissue and converting to equivalents of the parent compound. High Performance Liquid Chromatography (HPLC) is an excellent method for the chemical separation of complex mixtures whose profiles afford estimation of biochemical pathways of metabolism. Compounds elute from the HPLC systems with characteristic retention times and can be collected in fractions that can then be graphitized for AMS measurement.more » Unknowns are identified by coelution with known standards and chemical tests that reveal functional groupings. Metabolites are quantified with the {sup 14}C signal. Thoroughly accounting for the carbon inventory in the LC solvents, ion-pairing agents, samples, and carriers adds some complexity to the analysis. In most cases the total carbon inventory is dominated by carrier. Baseline background and stability need to be carefully monitored. Limits of quantitation near 10 amol of {sup 14}C per HPLC fraction are typically achieved. Baselines are maintained by limiting injected {sup 14}C activity <0.17 Bq (4.5 pCi) on the HPLC column.« less

New approaches with two cyano columns to the separation of acetaminophen, phenylephrine, chlorpheniramine and related compounds.

PubMed

Olmo, B; García, A; Marín, A; Barbas, C

2005-03-25

The development of new pharmaceutical forms with classical active compounds generates new analytical problems. That is the case of sugar-free sachets of cough-cold products containing acetaminophen, phenylephrine hydrochloride and chlorpheniramine maleate. Two cyanopropyl stationary phases have been employed to tackle the problem. The Discovery cyanopropyl (SUPELCO) column permitted the separation of the three actives, maleate and excipients (mainly saccharine and orange flavour) with a constant proportion of aqueous/ organic solvent (95:5, v/v) and a pH gradient from 7.5 to 2. The run lasted 14 min. This technique avoids many problems related to baseline shifts with classical organic solvent gradients and opens great possibilities to modify selectivity not generally used in reversed phase HPLC. On the other hand, the Agilent Zorbax SB-CN column with a different retention profile permitted us to separate not only the three actives and the excipients but also the three known related compounds: 4-aminophenol, 4-chloracetanilide and 4-nitrophenol in an isocratic method with a run time under 30 min. This method was validated following ICH guidelines and validation parameters showed that it could be employed as stability-indicating method for this pharmaceutical form.

Flow separation on flapping and rotating profiles with spanwise gradients.

PubMed

Wong, J G; laBastide, B P; Rival, D E

2017-02-15

The growth of leading-edge vortices (LEV) on analogous flapping and rotating profiles has been investigated experimentally. Three time-varying cases were considered: a two-dimensional reference case with a spanwise-uniform angle-of-attack variation α; a case with increasing α towards the profile tip (similar to flapping flyers); and a case with increasing α towards the profile root (similar to rotor blades experiencing an axial gust). It has been shown that the time-varying spanwise angle-of-attack gradient produces a vorticity gradient, which, in combination with spanwise flow, results in a redistribution of circulation along the profile. Specifically, when replicating the angle-of-attack gradient characteristic of a rotor experiencing an axial gust, the spanwise-vorticity gradient is aligned such that circulation increases within the measurement domain. This in turn increases the local LEV growth rate, which is suggestive of force augmentation on the blade. Reversing the relative alignment of the spanwise-vorticity gradient and spanwise flow, thereby replicating that arrangement found in a flapping flyer, was found to reduce local circulation. From this, we can conclude that spanwise flow can be arranged to vary LEV growth to prolong lift augmentation and reduce the unsteadiness of cyclic loads.

A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2-deoxyglucose in human plasma.

PubMed

Gounder, Murugesan K; Lin, Hongxia; Stein, Mark; Goodin, Susan; Bertino, Joseph R; Kong, Ah-Ng Tony; DiPaola, Robert S

2012-05-01

2-Deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate-boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45 min. The analytes were separated on a YMC ODS C₁₈ reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425 nm. The 2-DG calibration curves were linear over the range of 0.63-300 µg/mL with a limit of detection of 0.5 µg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8%, and the accuracy ranged from 86.8 to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors. Copyright © 2011 John Wiley & Sons, Ltd.

Development and validation of a stability-indicating RP-HPLC method for assay of betamethasone and estimation of its related compounds.

PubMed

Fu, Qiang; Shou, Minshan; Chien, Dwight; Markovich, Robert; Rustum, Abu M

2010-02-05

Betamethasone (9alpha-fluoro-16beta-methylprednisolone) is one of the members of the corticosteriod family of active pharmaceutical ingredient (API), which is widely used as an anti-inflammatory agent and also as a starting material to manufacture various esters of betamethasone. A stability-indicating reverse-phase high performance liquid chromatography (RP-HPLC) method has been developed and validated which can separate and accurately quantitate low levels of 26 betamethasone related compounds. The stability-indicating capability of the method was demonstrated through adequate separation of all potential betamethasone related compounds from betamethasone and also from each other that are present in aged and stress degraded betamethasone stability samples. Chromatographic separation of betamethasone and its related compounds was achieved by using a gradient elution at a flow rate of 1.0mL/min on a ACE 3 C18 column (150mmx4.6mm, 3microm particle size, 100A pore size) at 40 degrees C. Mobile phase A of the gradient was 0.1% methanesulfonic acid in aqueous solution and mobile phase B was a mixture of tert-butanol and 1,4-dioxane (7:93, v/v). UV detection at 254nm was employed to monitor the analytes. For betamethasone 21-aldehyde, the QL and DL were 0.02% and 0.01% respectively. For betamethasone and the rest of the betamethasone related compounds, the QL and DL were 0.05% and 0.02%. The precision of betamethasone assay is 0.6% and the accuracy of betamethasone assay ranged from 98.1% to 99.9%.

Separation and quantitative determination of 6α-hydroxycortisol and 6β-hydroxycortisol in human urine by high-performance liquid chromatography with ultraviolet absorption detection.

PubMed

Shibasaki, Hiromi; Okamoto, Sawako; Inoue, Risako; Okita, Misato; Yokokawa, Akitomo; Furuta, Takashi

2012-03-01

The present study developed an high-performance liquid chromatography (HPLC) method for the simultaneous determination of urinary metabolites of endogenous cortisol, 6α-hydroxycortisol (6α-OHF) and 6β-hydroxycortisol (6β-OHF), in human urine, using 6α-hydroxycorticosterone as internal standard. 6α-OHF and 6β-OHF were extracted from urine with ethyl acetate by using a Sep-Pak C(18) plus cartridge. Separation of the stereoisomers was achieved on a reversed-phase hybrid column by a gradient elution of (A) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and (B) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH/acetonitrile (2:3, v/v). 6α-OHF and 6β-OHF were well separated on an XTerra MS C(18) 5 μm column using two types of stepwise gradient elution program (programs 2 and 3). Resolutions of 6α-OHF and 6β-OHF were Rs = 4.41 for program 2 and Rs = 4.60 for program 3. The analysis was performed within 23~26 min, monitored by UV absorbance at 239 nm. The lower limits of detection of 6α-OHF and 6β-OHF were 0.80 ng per injection (s/n = ca. 8), and the lower limits of quantification were 5.02 ng/ml for 6α-OHF and 41.08 ng/ml for 6β-OHF, respectively. The within-day reproducibilities in the amounts of 6α-OHF and 6β-OHF determined were in good agreement with the actual amounts added, the relative errors being -5.37% and -3.73% (gradient 2) and -5.69% and -3.96% (gradient 3) for both 6α-OHF and 6β-OHF, respectively. The inter-assay precisions (RSDs) for 6α-OHF and 6β-OHF were less than 1.99% (gradient 2) and 2.61% (gradient 3), respectively. The present HPLC method was applied to the measurement of 6α-OHF and 6β-OHF in urine to evaluate the time courses of 6α-hydroxylation and 6β-hydroxylation clearances of cortisol during 40 days for phenotyping CYP3A in a healthy subject.

An on-line SPE-HPLC method for effective sample preconcentration and determination of fenoxycarb and cis, trans-permethrin in surface waters.

PubMed

Šatínský, Dalibor; Naibrtová, Linda; Fernández-Ramos, Carolina; Solich, Petr

2015-09-01

A new on-line SPE-HPLC method using fused-core columns for on-line solid phase extraction and large volume sample injection for increasing the sensitivity of detection was developed for the determination of insecticides fenoxycarb and cis-, trans-permethrin in surface waters. The separation was carried out on fused-core column Phenyl-Hexyl (100×4.6 mm), particle size 2.7 µm with mobile phase acetonitrile:water in gradient mode at flow rate 1.0 mL min(-1), column temperature 45°C. Large volume sample injection (1500 µL) to the extraction dimension using short precolumn Ascentis Express RP C-18 (5×4.6 mm); fused-core particle size 2.7 µm allowed effective sample preconcentration and efficient ballast sample matrix removal. The washing mobile phase consisting of a mixture of acetonitrile:water; 30:70, (v/v) was pumped at flow rate of 0.5 mL min(-1) through the extraction precolumn to the waste. Time of the valve switch for transferring the preconcentrated sample zone from the extraction to the separation column was set at 3rd min. Elution of preconcentrated insecticides from the extraction precolumn and separation on the analytical column was performed in gradient mode. Linear gradient elution started from 40% of acetonitrile at time of valve switch from SPE column (3rd min) to 95% of acetonitrile at 7th min. Synthetic dye sudan I was chosen as an internal standard. UV detection at wavelength 225 nm was used and the method reached the limits of detection (LOD) at ng mL(-1) levels for both insecticides. The method showing on-line sample pretreatment and preconcentration with highly sensitive determination of insecticides was applied for monitoring of fenoxycarb and both permethrin isomers in different surface water samples in Czech Republic. The time of whole analysis including on-line extraction, interferences removal, chromatography separation and system equilibration was less than 8 min. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitation of chlorophylls and 22 of their colored degradation products in culinary aromatic herbs by HPLC-DAD-MS and correlation with color changes during the dehydration process.

PubMed

Lafeuille, Jean-Louis; Lefèvre, Stéphane; Lebuhotel, Julie

2014-02-26

Chlorophylls and their green and olive-brown derivatives were successfully separated from culinary herb extracts by HPLC with photodiode-array and mass spectrometry detection. The method involved a ternary gradient elution and reverse-phase separation conditions capable of resolving 24 different pigments (2 chlorophylls and 22 of their derivatives) of different polarities within 28 min. The method was applied to monitor color changes in 50 samples of culinary aromatic herbs subjected to five different drying treatments. Of the 24 pigments, 14 were key to understanding the differences between the primary degradation pathways of chlorophyll a and chlorophyll b in culinary herbs during drying processes. A color degradation ladder based on the total molar percentage of all the remaining green pigments was also proposed as a tool to measure the impact of drying treatments on aromatic herb visual aspects.

A thin film degradation study of a fluorinated polyether liquid lubricant using an HPLC method

NASA Technical Reports Server (NTRS)

Morales, W.

1986-01-01

A High Pressure Liquid Chromatography (HPLC) separation method was developed to study and analyze a fluorinated polyether fluid which is promising liquid lubricant for future applications. This HPLC separation method was used in a preliminary study investigating the catalytic effect of various metal, metal alloy, and ceramic engineering materials on the degradation of this fluid in a dry air atmosphere at 345 C. Using a 440 C stainless steel as a reference catalytic material it was found that a titanium alloy and a chromium plated material degraded the fluorinated polyether fluid substantially more than the reference material.

Identification of candidate amino acids involved in the formation of pink-red pigments in onion (Allium cepa L.) juice and separation by HPLC.

PubMed

Lee, Eun Jin; Yoo, Kil Sun; Patil, Bhimanagouda S

2010-10-01

The formation of pink-red pigments ("pinking") by various amino acids was investigated by reacting amino acids with compounds present in onion juice. The unknown pink-red pigments were generated and separated using high-performance liquid chromatography (HPLC) and a diode array detector (DAD) in the range of 200 to 700 nm. To generate pink-red pigments, we developed several reaction systems using garlic alliinase, purified 1-propenyl-L-cysteine sulfoxide (1-PeCSO), onion thiosulfinate, natural onion juice, and 21 free amino acids. The compound 1-PeCSO was a key compound associated with pinking in the presence of both the alliinase and amino acids. Numerous naturally occurring pink-red pigments were detected and separated from pink onion juice using the HPLC-DAD system at 515 nm. Most free amino acids, with the exceptions of histidine, serine, and cysteine, formed various pink-red pigments when reacted with onion thiosulfinate. This observation indicated that onion pinking is caused not by a single pigment, but by many. Furthermore, more than one color compound could be produced from a single amino acid; this explains, in part, why there were many pink-red compound peaks in the chromatogram of discolored natural onion juice. We presumed that the complexity of the pink-red pigments was due to the involvement of more than 21 natural amino acids as well as several derivatives of the color products produced from each amino acid. We observed that the pinking process in onion juice is very similar to that of the greening process in crushed garlic, emphasizing that both thiosulfinate from flavor precursors and free amino acids are absolutely required for the discoloration.

An optimized high-performance liquid chromatography (HPLC) method for benzoylmesaconine determination in Radix Aconiti Lateralis Preparata (Fuzi, aconite roots) and its products

PubMed Central

Xie, Ying; Zhou, Hua; Wong, Yuen Fan; Liu, Zhongqiu; Xu, Hongxi; Jiang, Zhihong; Liu, Liang

2008-01-01

Background Benzoylmesaconine (BMA) is the main Aconitum alkaloid in Radix Aconiti Lateralis Preparata (Fuzi, aconite roots) with potent pharmacological activities, such as analgesia and anti-inflammation. The present study developed a simple and reliable method using BMA as a marker compound for the quality control of processed aconite roots and their products. Methods After extraction, a high-performance liquid chromatography (HPLC) determination of BMA was conducted on a RP-C18 column by gradient elution with acetonitrile and aqueous phase, containing 0.1% phosphoric acid adjusted with triethylamine to pH 3.0. Results A distinct peak profile was obtained and separation of BMA was achieved. Method validation showed that the relative standard deviations (RSDs) of the precision of BMA in all intra-day and inter-day assays were less than 1.36%, and that the average recovery rate was 96.95%. Quantitative analysis of BMA showed that the content of BMA varied significantly in processed aconite roots and their products. Conclusion This HPLC method using BMA as a marker compound is applicable to the quality control of processed aconite roots and their products. PMID:18513409

Optimization of Robust HPLC Method for Quantitation of Ambroxol Hydrochloride and Roxithromycin Using a DoE Approach.

PubMed

Patel, Rashmin B; Patel, Nilay M; Patel, Mrunali R; Solanki, Ajay B

2017-03-01

The aim of this work was to develop and optimize a robust HPLC method for the separation and quantitation of ambroxol hydrochloride and roxithromycin utilizing Design of Experiment (DoE) approach. The Plackett-Burman design was used to assess the impact of independent variables (concentration of organic phase, mobile phase pH, flow rate and column temperature) on peak resolution, USP tailing and number of plates. A central composite design was utilized to evaluate the main, interaction, and quadratic effects of independent variables on the selected dependent variables. The optimized HPLC method was validated based on ICH Q2R1 guideline and was used to separate and quantify ambroxol hydrochloride and roxithromycin in tablet formulations. The findings showed that DoE approach could be effectively applied to optimize a robust HPLC method for quantification of ambroxol hydrochloride and roxithromycin in tablet formulations. Statistical comparison between results of proposed and reported HPLC method revealed no significant difference; indicating the ability of proposed HPLC method for analysis of ambroxol hydrochloride and roxithromycin in pharmaceutical formulations. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

HPLC Separation of Vitamin E and Its Oxidation Products and Effects of Oxidized Tocotrienols on the Viability of MCF-7 Breast Cancer Cells in Vitro.

PubMed

Drotleff, Astrid M; Büsing, Anne; Willenberg, Ina; Empl, Michael T; Steinberg, Pablo; Ternes, Waldemar

2015-10-14

Tocotrienols, a vitamin E subgroup, exert potent anticancer effects, but easily degrade due to oxidation. Eight vitamin E reference compounds, α-, β-, γ-, or δ-tocopherols or -tocotrienols, were thermally oxidized in n-hexane. The corresponding predominantly dimeric oxidation products were separated from the parent compounds by diol-modified normal-phase HPLC-UV and characterized by mass spectroscopy. The composition of test compounds, that is, α-tocotrienol, γ-tocotrienol, or palm tocotrienol-rich fraction (TRF), before and after thermal oxidation was determined by HPLC-DAD, and MCF-7 cells were treated with both nonoxidized and oxidized test compounds for 72 h. Whereas all nonoxidized test compounds (0-100 μM) led to dose-dependent decreases in cell viability, equimolar oxidized α-tocotrienol had a weaker effect, and oxidized TRF had no such effect. However, the IC50 value of oxidized γ-tocotrienol was lower (85 μM) than that of nonoxidized γ-tocotrienol (134 μM), thereby suggesting that γ-tocotrienol oxidation products are able to reduce tumor cell viability in vitro.

Chiral HPLC for a study of the optical purity of new liquid crystalline materials derived from lactic acid

NASA Astrophysics Data System (ADS)

Vojtylová, T.; Kašpar, M.; Hamplová, V.; Novotná, V.; Sýkora, D.

2014-08-01

New liquid crystalline (LC) materials were prepared by derivatization of lactic acid. First compound possesses the lactic acid unit as the only chiral center and the second group of LC materials contains two chiral centers. Mesomorphic properties of both the newly synthesized LC materials were studied and the presence of the SmA*-SmC* or exhibit the twist grain boundary (TGB) phases, namely TGBA and TGBC, in a wide range of temperatures down to the room temperature was established. The potential of high-performance liquid chromatography (HPLC) applying chiral stationary phases to separate enantiomers or diastereoisomers of the synthesized LC compounds was evaluated. Two different brands of commercial chiral sorbents, Lux Amylose-2 and Chiralpak AD-3, both based on modified silica with derivatized polysaccharide, were employed in the development of separation procedures. The optimized chiral HPLC method provided a baseline separation of the individual enantiomers for the LC material containing one chiral center. In the case of the more complex compound with two asymmetric carbon atoms, where four isomers exist, partial separation was reached only using the current chiral HPLC.

Application of linear pH gradients for the modeling of ion exchange chromatography: Separation of monoclonal antibody monomer from aggregates.

PubMed

Kluters, Simon; Wittkopp, Felix; Jöhnck, Matthias; Frech, Christian

2016-02-01

The mobile phase pH is a key parameter of every ion exchange chromatography process. However, mechanistic insights into the pH influence on the ion exchange chromatography equilibrium are rare. This work describes a mechanistic model capturing salt and pH influence in ion exchange chromatography. The pH dependence of the characteristic protein charge and the equilibrium constant is introduced to the steric mass action model based on a protein net charge model considering the number of amino acids interacting with the stationary phase. This allows the description of the adsorption equilibrium of the chromatographed proteins as a function of pH. The model parameters were determined for a monoclonal antibody monomer, dimer, and a higher aggregated species based on a manageable set of pH gradient experiments. Without further modification of the model parameters the transfer to salt gradient elution at fixed pH is demonstrated. A lumped rate model was used to predict the separation of the monoclonal antibody monomer/aggregate mixture in pH gradient elution and for a pH step elution procedure-also at increased protein loadings up to 48 g/L packed resin. The presented model combines both salt and pH influence and may be useful for the development and deeper understanding of an ion exchange chromatography separation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography.

PubMed

Cao, Xueli; Wang, Qiaoe; Li, Yan; Bai, Ge; Ren, Hong; Xu, Chunming; Ito, Yoichiro

2011-03-01

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, ¹HNMR and ¹³CNMR. Copyright © 2011 Elsevier B.V. All rights reserved.

Isolation and purification of series bioactive components from Hypericum Perforatum L. by high-speed counter-current chromatography

PubMed Central

Cao, Xueli; Wang, Qiaoe; Li, Yan; Bai, Ge; Ren, Hong; Ito, Yiochiro

2011-01-01

High-speed counter-current chromatography (HSCCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate–water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR. PMID:21306961

Discrimination of red and white rice bran from Indonesia using HPLC fingerprint analysis combined with chemometrics.

PubMed

Sabir, Aryani; Rafi, Mohamad; Darusman, Latifah K

2017-04-15

HPLC fingerprint analysis combined with chemometrics was developed to discriminate between the red and the white rice bran grown in Indonesia. The major component in rice bran is γ-oryzanol which consisted of 4 main compounds, namely cycloartenol ferulate, cyclobranol ferulate, campesterol ferulate and β-sitosterol ferulate. Separation of these four compounds along with other compounds was performed using C18 and methanol-acetonitrile with gradient elution system. By using these intensity variations, principal component and discriminant analysis were performed to discriminate the two samples. Discriminant analysis was successfully discriminated the red from the white rice bran with predictive ability of the model showed a satisfactory classification for the test samples. The results of this study indicated that the developed method was suitable as quality control method for rice bran in terms of identification and discrimination of the red and the white rice bran. Copyright © 2016 Elsevier Ltd. All rights reserved.

An Efficient Method for the Preparative Isolation and Purification of Flavonoid Glycosides and Caffeoylquinic Acid Derivatives from Leaves of Lonicera japonica Thunb. Using High Speed Counter-Current Chromatography (HSCCC) and Prep-HPLC Guided by DPPH-HPLC Experiments.

PubMed

Wang, Daijie; Du, Ning; Wen, Lei; Zhu, Heng; Liu, Feng; Wang, Xiao; Du, Jinhua; Li, Shengbo

2017-02-02

In this work, the n-butanol extract from leaves of Lonicera japonica Thunb. (L. japonica) was reacted with DPPH and subjected to a HPLC analysis for the guided screening antioxidants (DPPH-HPLC experiments). Then, nine antioxidants, including flavonoid glycosides and caffeoylquinic acid derivatives, were isolated and purified from leaves of L. japonica using high speed counter-current chromatography (HSCCC) and prep-HPLC. The n-butanol extract was firstly isolated by HSCCC using methyl tert-butyl ether/n-butanol/acetonitrile/water (0.5% acetic acid) (2:2:1:5, v/v), yielding five fractions F1, F2 (rhoifolin), F3 (luteoloside), F4 and F5 (collected from the column after the separation). The sub-fractions F1, F4 and F5 were successfully separated by prep-HPLC. Finally, nine compounds, including chlorogenic acid (1), lonicerin (2), rutin (3), rhoifolin (4), luteoloside (5), 3,4-Odicaffeoylquinic acid (6), hyperoside (7), 3,5-O-dicaffeoylquinic acid (8), and 4,5-O-dicaffeoylquinic acid (9) were obtained, respectively, with the purities over 94% as determined by HPLC. The structures were identified by electrospray ionization mass spectrometry (ESI-MS), 1H- and 13C-NMR. Antioxidant activities were tested, and the isolated compounds showed strong antioxidant activities.

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

PubMed Central

Yang, Yi; Zhao, Yongxin; Gu, Dongyu; Ayupbek, Amatjan; Huang, Yun; Dou, Jun; Ito, Yoichiro; Zhang, Tianyou; Aisa, Haji Akber

2010-01-01

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4′-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, 1H NMR and 13C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC. PMID:21494318

Development and validation of a HPLC-UV-ESI-MS method for the simultaneous quantitation of ten bioactive compounds in Dahuang Fuzi Tang.

PubMed

Guo, Hui; Li, Huan; Liu, Xiao; Cai, Hao; Wu, Li; Cai, Bao-Chang

2014-12-01

To develop a high performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) and ultraviolet (UV) detector method for the acid-alkaline simultaneous determination of ten bioactive compounds, and analyze the effect of compatible medicinal plants on the concentration of components in Dahuang Fuzi Tang (DFT). The chromatographic separation was performed on a Hypersil BDS C18 analytical column by gradient elution with acetonitrile and formate buffer (containing 0.15% formic acid, V/V) at 25 °C with a flow rate of 1.0 mL·min(-1) and UV detection at 280 nm. Four of the ten compounds in DFT were identified and their MS fragments were elucidated by HPLC-ESI-MS, and the contents of the six compounds were determined by HPLC-UV. All calibration curves showed good linear regression (r(2) ≥ 0.9990). The limits of detection and limits of quantification were 0.021-0.155 -g·mL(-1) and 0.076-0.520 -g·mL(-1), respectively. Overall precision RSD (intra-day and inter-day) were less than 2.96%, and the average recoveries were 98.35%-101.45%, with RSD ranging from 1.54% to 3.01% for the analytes. The developed method can be applied for the quality control and provide analytical evidence on the chemical basis and combinational principles of DFT. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

HPLC separation of human serum albumin isoforms based on their isoelectric points

PubMed Central

Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A.; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

2014-01-01

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA–SHg+), HSA with Cys34 oxidized to sulfenic acid (HSA–SOH) and HSA oxidized to sulfinate anion (HSA–SO2−) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3–585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA PMID:24316526

HPLC separation of human serum albumin isoforms based on their isoelectric points.

PubMed

Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

2014-01-01

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA. Copyright © 2013 Elsevier B.V. All rights reserved.

A validated HPLC-PDA method for identification and quantification of two bioactive alkaloids, ephedrine and cryptolepine, in different Sida species.

PubMed

Chatterjee, Arnab; Kumar, Satyanshu; Chattopadhyay, Sunil K

2013-12-01

A simple, rapid, accurate and reproducible reverse-phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP-18 X-terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10-250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra-day and inter-day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species. Copyright © 2013 John Wiley & Sons, Ltd.

Development and Validation of an HPLC Method for Simultaneous Determination of Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol Hydrochloride in Pharmaceutical Formulations.

PubMed

Chellini, Paula R; Lages, Eduardo B; Franco, Pedro H C; Nogueira, Fernando H A; César, Isabela C; Pianetti, Gerson A

2015-01-01

Tuberculosis treatment consists of a fixed dose combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PYZ), and ethambutol hydrochloride (EMB). The combined treatment using various drugs is necessary for patient curing, without recrudescence, and for prevention of drug-resistant mutants, which may occur during treatment. An HPLC-diode array detector (DAD) method for the simultaneous determination of RIF, INH, PYZ, and EMB in fixed dose combination tablets was developed and validated. Chromatographic experiments were performed on an Agilent 1200 HPLC system, and the separation was carried out on a Purospher STAR RP18e (250×4.6 mm id, 5 μm, Merck) analytical column. Gradient elution was carried out with a mobile phase of 20 mM monobasic sodium phosphate buffer with 0.2% triethylamine (pH 7.0) and acetonitrile at a flow rate of 1.5 mL/min. The total run time was 12 min, and the re-equilibration time was 5 min. EMB detection was performed at 210 nm, and RIF, INH, and PYZ were detected at 238 nm, using a DAD. The method proved to be specific, linear (r2>0.99), precise (RSD<2%), accurate, and robust and may be applied to the QC analysis of pharmaceutical formulations.

Unsteady separation and vortex shedding from a laminar separation bubble over a bluff body

NASA Astrophysics Data System (ADS)

Das, S. P.; Srinivasan, U.; Arakeri, J. H.

2013-07-01

Boundary layers are subject to favorable and adverse pressure gradients because of both the temporal and spatial components of the pressure gradient. The adverse pressure gradient may cause the flow to separate. In a closed loop unsteady tunnel we have studied the initiation of separation in unsteady flow past a constriction (bluff body) in a channel. We have proposed two important scalings for the time when boundary layer separates. One is based on the local pressure gradient and the other is a convective time scale based on boundary layer parameters. The flow visualization using a dye injection technique shows the flow structure past the body. Nondimensional shedding frequency (Strouhal number) is calculated based on boundary layer and momentum thicknesses. Strouhal number based on the momentum thickness shows a close agreement with that for flat plate and circular cylinder.

[Quantitative analysis of nucleosides in four Cordyceps genus by HPLC].

PubMed

Qian, Zheng-Ming; Li, Wen-Qing; Wang, Chuan-Xi; Zhou, Miao-Xia; Sun, Min-Tian; Gao, Hao; Li, Wen-Jia

2016-07-01

To compare the main nucleosides in Cordyceps genus herbs (C. sinensis, C. millitaris, Hirsutella sinensis and C. sobolifera), an HPLC method for simultaneous determination of uridine, inosine, guanosine, adenosine and cordycepine in Cordyceps genus herbs was developed. The sample was extracted with 0.5% phosphoric acid solution to prepare test solution. The separation was performed on a Zorbax SB-Aq (4.6 mm×150 mm, 5 μm) column with gradient elution by 0.04 mol•L⁻¹ potassium dihydrogen phosphate solution and acetonitrile, column temperature 30 ℃,flow rate 0.8 mL•min⁻¹,and detection wavelength 260 nm. The content of nucleosides in four Cordyceps genus herbs was evaluated by fingerprint analysis and hierarchical cluster analysis (HCA). The calibration curves of five nucleosides showed good linear regression (r>0.99) and the average recoveries were between 95.0% and 105.0%. The contents of the five nucleosides in the four Cordyceps genus herbs were different and could be obviously distinguished by HCA. The fingerprint analysis result showed that the similarity between C. sinensis and the others was less than 0.9. The method was accurate and reliable, which can be used for quality control of Cordyceps genus herbs. Copyright© by the Chinese Pharmaceutical Association.

Measurement of the adhesion force between particles for high gradient magnetic separation of pneumatic conveyed powder products

NASA Astrophysics Data System (ADS)

Senkawa, K.; Nakai, Y.; Mishima, F.; Akiyama, Y.; Nishijima, S.

2011-11-01

In the industrial plants such as foods, medicines or industrial materials, there are big amount of issues on contamination by metallic wear debris originated from pipes of manufacturing lines. In this study, we developed a high gradient magnetic separation system (HGMS) under the dry process by using superconducting magnet to remove the ferromagnetic particles. One of the major problems of dry HGMS systems is, however, the blockage of magnetic filter caused by particle coagulation or deposition. In order to actualize the magnetic separation without blockage, we introduced pneumatic conveyance system as a new method to feed the powder. It is important to increase the drag force acting on the sufficiently dispersed particles, which require strong magnetic fields. To generate the strong magnetic fields, HGMS technique was examined which consists of a magnetic filter and a superconducting solenoid magnet. As a result of the magnetic separation experiment, it was shown that the separation efficiency changes due to the difference of the cohesive property of the particles. On the basis of the result, the adhesion force which acts between the ferromagnetic particles and the medium particles used for the magnetic separation was measured by Atomic Force Microscope (AFM), and cohesion of particles was studied from the aspect of interparticle interaction. We assessed a suitable flow velocity for magnetic separation according to the cohesive property of each particle based on the result.

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 2: Computer controlled pH gradients in the presence of urea provide improved separation of proteins: Stability influenced anion and cation exchange chromatography.

PubMed

Hirsh, Allen G; Tsonev, Latchezar I

2017-04-28

This paper details the use of a method of creating controlled pH gradients (pISep) to improve the separation of protein isoforms on ion exchange (IEX) stationary phases in the presence of various isocratic levels of urea. The pISep technology enables the development of computer controlled pH gradients on both cationic (CEX) and anionic (AEX) IEX stationary phases over the very wide pH range from 2 to 12. In pISep, titration curves generated by proportional mixing of the acidic and basic pISep working buffers alone, or in the presence of non-buffering solutes such as the neutral salt NaCl (0-1M), polar organics such as urea (0-8M) or acetonitrile (0-80 Vol%), can be fitted with high fidelity using high order polynomials which, in turn allows construction of a mathematical manifold %A (% acidic pISep buffer) vs. pH vs. [non-buffering solute], permitting precise computer control of pH and the non-buffering solute concentration allowing formation of dual uncoupled liquid chromatographic (LC) gradients of arbitrary shape (Hirsh and Tsonev, 2012 [1]). The separation of protein isoforms examined in this paper by use of such pH gradients in the presence of urea demonstrates the fractionation power of a true single step two dimensional liquid chromatography which we denote as Stability-Influenced Ion Exchange Chromatography (SIIEX). We present evidence that SIIEX is capable of increasing the resolution of protein isoforms difficult to separate by ordinary pH gradient IEX, and potentially simplifying the development of laboratory and production purification strategies involving on-column simultaneous pH and urea unfolding or refolding of targeted proteins. We model some of the physics implied by the dynamics of the observed protein fractionations as a function of both urea concentration and pH assuming that urea-induced native state unfolding competes with native state electrostatic interaction binding to an IEX stationary phase. Implications for in vivo protein

Simultaneous determination of celecoxib, hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid chromatography with ultraviolet absorbance detection.

PubMed

Störmer, Elke; Bauer, Steffen; Kirchheiner, Julia; Brockmöller, Jürgen; Roots, Ivar

2003-01-05

A new HPLC method for the simultaneous determination of celecoxib, carboxycelecoxib and hydroxycelecoxib in human plasma samples has been developed. Following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HLPC (C(18)) and quantified using UV detection at 254 nm. The method was linear over the concentration range 10-500 ng/ml. The intra-assay variability for the three analytes ranged from 4.0 to 12.6% and the inter-assay variability from 4.9 to 14.2%. The achieved limits of quantitation (LOQ) of 10 ng/ml for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib.

Stereo transparency and the disparity gradient limit

NASA Technical Reports Server (NTRS)

McKee, Suzanne P.; Verghese, Preeti

2002-01-01

Several studies (Vision Research 15 (1975) 583; Perception 9 (1980) 671) have shown that binocular fusion is limited by the disparity gradient (disparity/distance) separating image points, rather than by their absolute disparity values. Points separated by a gradient >1 appear diplopic. These results are sometimes interpreted as a constraint on human stereo matching, rather than a constraint on fusion. Here we have used psychophysical measurements on stereo transparency to show that human stereo matching is not constrained by a gradient of 1. We created transparent surfaces composed of many pairs of dots, in which each member of a pair was assigned a disparity equal and opposite to the disparity of the other member. For example, each pair could be composed of one dot with a crossed disparity of 6' and the other with uncrossed disparity of 6', vertically separated by a parametrically varied distance. When the vertical separation between the paired dots was small, the disparity gradient for each pair was very steep. Nevertheless, these opponent-disparity dot pairs produced a striking appearance of two transparent surfaces for disparity gradients ranging between 0.5 and 3. The apparent depth separating the two transparent planes was correctly matched to an equivalent disparity defined by two opaque surfaces. A test target presented between the two transparent planes was easily detected, indicating robust segregation of the disparities associated with the paired dots into two transparent surfaces with few mismatches in the target plane. Our simulations using the Tsai-Victor model show that the response profiles produced by scaled disparity-energy mechanisms can account for many of our results on the transparency generated by steep gradients.

A novel tertiary prep-HPLC method for the isolation of single amino acids for AMS-radiocarbon measurement.

PubMed

Fernandes, Ricardo; Koudelka, Tomas; Tholey, Andreas; Dreves, Alexander

2017-07-15

AMS-radiocarbon measurements of amino acids can potentially provide more reliable radiocarbon dates than bulk collagen analysis. Nonetheless, the applicability of such an approach is often limited by the low-throughput of existing isolation methods and difficulties in determining the contamination introduced during the separation process. A novel tertiary prep-HPLC amino acid isolation method was developed that relies on the combustion of eluted material without requiring any additional chemical steps. Amino acid separation was carried out using a gradient mix of pure water and phosphoric acid with an acetonitrile step in-between runs to remove hydrophobic molecules from the separation column. The amount of contaminant carbon and its 14 C content were determined from two-point measurements of collagen samples of known 14 C content. The amount of foreign carbon due to the isolation process was estimated at 4±1μg and its 14 C content was 0.43±0.01 F 14 C. Radiocarbon values corrected for carbon contamination have only a minor increase in uncertainties. For Holocene samples, this corresponds to an added uncertainty typically smaller than 10 14 Cyears. The developed method can be added to routine AMS measurements without implying significant operational changes and offers a level of measurement uncertainty that is suitable for many archaeological, ecological, environmental, and biological applications. Copyright © 2017. Published by Elsevier B.V.

Development of high gradient magnetic separation system for removing the metallic wear debris to be present in highly viscous fluid

NASA Astrophysics Data System (ADS)

Hayashi, S.; Mishima, F.; Akiyama, Y.; Nishijima, S.

2010-11-01

In the industrial plants processing highly viscous fluid such as foods or industrial materials, there is an issue of contamination by metallic wear debris originating from pipe of manufacturing line. It is necessary to remove the metallic wear debris in highly viscous fluid, since these debris causes quality loss. In this study, we developed a high gradient magnetic separation system by using superconducting magnet to remove the metallic wear debris. The particle trajectory simulation and the magnetic separation experiment were conducted with polyvinyl alcohol as a model material. As a result, ca. 100% and 92.2% of the separation efficiency was achieved respectively for the highly viscous fluid of 1 Pa s and 6 Pa s in viscosity, with 14 and 30 mesh magnetic filters.

The application of pulse field gradient (PFG) NMR methods to characterize the efficiency of separation of water-in-crude oil emulsions.

PubMed

Marques, Debora Salomon; Sørland, Geir; Less, Simone; Vilagines, Regis

2018-02-15

Demulsification of water-in-crude oil emulsions is an essential and sometimes challenging procedure for crude oil processing facilities. Pulse field gradient (PFG) NMR techniques are known to monitor the dynamics of emulsion separation. This method has limitations that restrict its application to some crude oils. A comprehensive methodology applicable to all types of crude oil regardless of its viscosity, without assumptions, and providing a large number of data with fast measurements, is proposed in this paper. The coalescence and sedimentation of unstable emulsions was observed through simultaneous measurements of the evolution of the brine profile and droplet size distribution (DSD). Measurements of emulsions after stabilization, with and without the contribution of the free water layer, revealed the residual emulsified water quantity and location in the sample. A new, faster approach to separate the oil and water overlapping T 2 relaxation signals was demonstrated on real water-in-crude oil emulsions, using the root mean square displacement (RMSD) measured with the spoiler recovery and a loop of 13-interval pulsed field gradient stimulated echo (PFGSTE) oneshot sequences. The residual water within the crude oils after separation was determined and used to quantify the efficiency of the demulsifier used. Copyright © 2017 Elsevier Inc. All rights reserved.

Performances of CN-columns for the analysis of γ-oryzanol and its p-coumarate and caffeate derivatives by normal phase HPLC and a validated method of quantitation.

PubMed

D'Ambrosio, Michele

2013-06-15

γ-Oryzanol is an important phytochemical used in pharmaceutical, alimentary and cosmetic preparations. The present article, for the first time, discloses the performances of NP-HPLC in separating γ-oryzanol components and develops a validated method for its routine quantification. The analysis is performed on a cyanopropyl bonded column using the hexane/MTBE gradient elution and UV detection at 325 nm. The method allows: the separation of steryl ferulate, p-coumarate and caffeate esters, the separation of cis- from trans-ferulate isomers, the splitting of steroid moieties into saturated and unsaturated at the side chain. The optimised method provides excellent linear response (R(2)=0.99997), high precision (RSD<1.0%) and satisfactory accuracy (R(∗)=70-86%). In conclusion, the established method presents the details of the procedure and the experimental conditions in order to achieve the required precision and instrumental accuracy. The method is fast and sensitive and it could be a suitable tool for quality assurance and determination of origin. Copyright © 2012 Elsevier Ltd. All rights reserved.

Analysis of the extracts of Isatis tinctoria by new analytical approaches of HPLC, MS and NMR.

PubMed

Zhou, Jue; Qu, Fan

2011-01-01

The methods of extraction, separation and analysis of alkaloids and indole glucosinolates (GLs) ofIsatis tinctoria were reviewed. Different analytical approaches such as High-pressure Liquid Chromatography (HPLC), Liquid Chromatography with Electrospray Ionization Mass Spectrometry (LC/ESI/MS), Electrospray Ionization Time-Of-Flight Mass Spectrometry (ESI-TOF-MS), and Nuclear Magnetic Resonance (NMR) were used to validate and identity of these constituents. These methods provide rapid separation, identification and quantitative measurements of alkaloids and GLs of Isatis tinctoria. By connection with different detectors to HPLC such as PDA, ELSD, ESI- and APCI-MS in positive and negative ion modes, complicated compounds could be detected with at least two independent detection modes. The molecular formula can be derived in a second step of ESI-TOF-MS data. But for some constituents, UV and MS cannot provide sufficient structure identification. After peak purification, NMR by semi-preparative HPLC can be used as a complementary method.

The Mysterious Death: An HPLC Lab Experiment. An Undergraduate Forensic Lab

ERIC Educational Resources Information Center

Beussman, Douglas J.

2007-01-01

A high-performance liquid chromatography (HPLC) laboratory experiment based on the separation of four prescription drugs (disopyramide, lidocaine, procainamide, and quinidine) is presented. The experiment is set within the forensic science context of the discovery of a patient's mysterious death where a drug overdose is suspected. Each lab group…

Characterization of chemical constituents in Rhodiola Crenulate by high-performance liquid chromatography coupled with Fourier-transform ion cyclotron resonance mass spectrometer (HPLC-FT-ICR MS).

PubMed

Han, Fei; Li, Yanting; Mao, Xinjuan; Xu, Rui; Yin, Ran

2016-05-01

In this work, an approach using high-performance liquid chromatography coupled with diode-array detection and Fourier-transform ion cyclotron resonance mass spectrometer (HPLC-FT-ICR MS) for the identification and profiling of chemical constituents in Rhodiola crenulata was developed for the first time. The chromatographic separation was achieved on an Inertsil ODS-3 column (150 mm × 4.6 mm,3 µm) using a gradient elution program, and the detection was performed on a Bruker Solarix 7.0 T mass spectrometer equipped with electrospray ionization source in both positive and negative modes. Under the optimized conditions, a total of 48 chemical compounds, including 26 alcohols and their glycosides, 12 flavonoids and their glycosides, 5 flavanols and gallic acid derivatives, 4 organic acids and 1 cyanogenic glycoside were identified or tentatively characterized. The results indicated that the developed HPLC-FT-ICR MS method with ultra-high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents in R. crenulata. And it provides a helpful chemical basis for further research on R. crenulata. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Insights into chromatographic separation using core-shell metal-organic frameworks: Size exclusion and polarity effects.

PubMed

Qin, Weiwei; Silvestre, Martin E; Kirschhöfer, Frank; Brenner-Weiss, Gerald; Franzreb, Matthias

2015-09-11

Porous metal-organic frameworks (MOFs) [Cu3(BTC)2(H2O)3]n (also known as HKUST-1; BTC, benzene-1,3,5-tricarboxylic acid) were synthesized as homogeneous shell onto carboxyl functionalized magnetic microparticles through a liquid phase epitaxy (LPE) process. The as-synthesized core-shell HKUST-1 magnetic microparticles composites were characterized by XRD and SEM, and used as stationary phase in high performance liquid chromatography (HPLC). The effects of the unique properties of MOFs onto the chromatographic performance are demonstrated by the experiments. First, remarkable separation of pyridine and bipyridine is achieved, although both molecules show a strong interaction between the Cu-ions in HKUST-1 and the nitrogen atoms in their heterocyles. The difference can be explained due to size exclusion of bipyridine from the well defined pore structure of crystalline HKUST-1. Second, the enormous variety of possible interactions of sample molecules with the metal ions and linkers within MOFs allows for specifically tailored solid phases for challenging separation tasks. For example, baseline separation of three chloroaniline (CLA) isomers tested can be achieved without the need for gradient elution modes. Along with the experimental HPLC runs, in-depth modelling with a recently developed chromatography modelling software (ChromX) was applied and proofs the software to be a powerful tool for exploring the separation potential of thin MOF films. The pore diffusivity of pyridine and CLA isomers within HKUST-1 are found to be around 2.3×10(-15)m(2)s(-1). While the affinity of HKUST-1 to the tested molecules strongly differs, the maximum capacities are in the same range, with 0.37molL(-1) for pyridine and 0.23molL(-1) for CLA isomers, corresponding to 4.0 and 2.5 molecules per MOF unit cell, respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative estimation of gymnemagenin in Gymnema sylvestre extract and its marketed formulations using the HPLC-ESI-MS/MS method.

PubMed

Kamble, Bhagyashree; Gupta, Ankur; Patil, Dada; Janrao, Shirish; Khatal, Laxman; Duraiswamy, B

2013-02-01

Gymnema sylvestre, with gymnemic acids as active pharmacological constituents, is a popular ayurvedic herb and has been used to treat diabetes, as a remedy for cough and as a diuretic. However, very few analytical methods are available for quality control of this herb and its marketed formulations. To develop and validate a new, rapid, sensitive and selective HPLC-ESI (electrospray ionisation)-MS/MS method for quantitative estimation of gymnemagenin in G. sylvestre and its marketed formulations. HPLC-ESI-MS/MS method using a multiple reactions monitoring mode was used for quantitation of gymnemagenin. Separation was carried out on a Luna C-18 column using gradient elution of water and methanol (with 0.1% formic acid and 0.3% ammonia). The developed method was validated as per International Conference on Harmonisation Guideline ICH-Q2B and found to be accurate, precise and linear over a relatively wide range of concentrations (5.280-305.920 ng/mL). Gymnemagenin contents were found from 0.056 ± 0.002 to 4.77 ± 0.59% w/w in G. sylvestre and its marketed formulations. The method established is simple, rapid, with high sample throughput, and can be used as a tool for quality control of G. sylvestre and its formulations. Copyright © 2012 John Wiley & Sons, Ltd.

Chromatographic Separations Using Solid-Phase Extraction Cartridges: Separation of Wine Phenolics

NASA Astrophysics Data System (ADS)

Brenneman, Charles A.; Ebeler, Susan E.

1999-12-01

We describe a simple laboratory experiment that demonstrates the principles of chromatographic separation using solid-phase extraction columns and red wine. By adjusting pH and mobile phase composition, the wine is separated into three fractions of differing polarity. The content of each fraction can be monitored by UV-vis spectroscopy. When the experiment is combined with experiments involving HPLC or GC separations, students gain a greater appreciation for and understanding of the highly automated instrumental systems currently available. In addition, they learn about the chemistry of polyphenolic compounds, which are present in many foods and beverages and which are receiving much attention for their potentially beneficial health effects.

G-index: A new metric to describe dynamic refractive index effects in HPLC absorbance detection.

PubMed

Kraiczek, Karsten G; Rozing, Gerard P; Zengerle, Roland

2018-09-01

High performance liquid chromatography (HPLC) with a solvent gradient and absorbance detection is one of the most widely used methods in analytical chemistry. The observed absorbance baseline is affected by the changes in the refractive index (RI) of the mobile phase. Near the limited of detection, this complicates peak quantitation. The general aspects of these RI-induced apparent absorbance effects are discussed. Two different detectors with fundamentally different optics and flow cell concepts, a variable-wavelength detector equipped with a conventional flow cell and a diode-array detector equipped with a liquid core waveguide flow cell, are compared with respect to their RI behavior. A simple method to separate static - partly unavoidable - RI effects from dynamic RI effects is presented. It is shown that the dynamic RI behavior of an absorbance detector can be well described using a single, relatively easy-to-determine metric called the G-index. The G-index is typically in the order of a few seconds and its sign depends on the optical flow cell concept. Copyright © 2018 Elsevier B.V. All rights reserved.

Separation of cells from the rat anterior pituitary gland

NASA Technical Reports Server (NTRS)

Hymer, Wesley C.; Hatfield, J. Michael

1983-01-01

Various techniques for separating the hormone-producing cell types from the rat anterior pituitary gland are examined. The purity, viability, and responsiveness of the separated cells depend on the physiological state of the donor, the tissue dissociation procedures, the staining technique used for identification of cell type, and the cell separation technique. The chamber-gradient setup and operation, the characteristics of the gradient materials, and the separated cell analysis of velocity sedimentation techniques (in particular Staput and Celsep) are described. Consideration is given to the various types of materials used in density gradient centrifugation and the operation of a gradient generating device. The use of electrophoresis to separate rat pituitary cells is discussed.

Enhanced fluidity liquid chromatography of inulin fructans using ternary solvent strength and selectivity gradients.

PubMed

Bennett, Raffeal; Olesik, Susan V

2018-01-25

The value of exploring selectivity and solvent strength ternary gradients in enhanced fluidity liquid chromatography (EFLC) is demonstrated for the separation of inulin-type fructans from chicory. Commercial binary pump systems for supercritical fluid chromatography only allow for the implementation of ternary solvent strength gradients which can be restrictive for the separation of polar polymeric analytes. In this work, a custom system was designed to extend the capability of EFLC to allow tuning of selectivity or solvent strength in ternary gradients. Gradient profiles were evaluated using the Berridge function (RF 1 ), normalized resolution product (NRP), and gradient peak capacity (P c ). Selectivity gradients provided the separation of more analytes over time. The RF 1 function showed favor to selectivity gradients with comparable P c to that of solvent strength gradients. NRP did not strongly correlate with P c or RF 1 score. EFLC with the hydrophilic interaction chromatography, HILIC, separation mode was successfully employed to separate up to 47 fructan analytes in less than 25 min using a selectivity gradient. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous determination of 15 phenolic constituents of Chinese black rice wine by HPLC-MS/MS with SPE.

PubMed

Wang, Yutang; Liu, Yuanyuan; Xiao, Chunxia; Liu, Laping; Hao, Miao; Wang, Jianguo; Liu, Xuebo

2014-06-01

This study established a new method for quantitative and qualitative determination of certain components in black rice wine, a traditional Chinese brewed wine. Specifically, we combined solid-phase extraction and high-performance liquid chromatography (HPLC) with triple quadrupole mass spectrometry (MS/MS) to determine 8 phenolic acids, 3 flavonols, and 4 anthocyanins in black rice wine. First, we clean samples with OASIS HLB cartridges and optimized extraction parameters. Next, we performed separation on a SHIM-PACK XR-ODS column (I.D. 3.0 mm × 75 mm, 2.2 μm particle size) with a gradient elution of 50% aqueous acetonitrile (V/V) and water, both containing 0.2% formic acid. We used multiple-reaction monitoring scanning for quantification, with switching electrospray ion source polarity between positive and negative modes in a single chromatographic run. We detected 15 phenolic compounds properly within 38 min under optimized conditions. Limits of detection ranged from 0.008 to 0.030 mg/L, and average recoveries ranged from 60.8 to 103.1% with relative standard deviation ≤8.6%. We validated the method and found it to be sensitive and reliable for quantifying phenolic compounds in rice wine matrices. This study developed a new, reliable HPLC-MS/MS method for simultaneous determination of 15 bioactive components in black rice wine. This method was validated and found to be sensitive and reliable for quantifying phenolic compounds in rice wine. © 2014 Institute of Food Technologists®

Comparison of various second-dimension gradient types in comprehensive two-dimensional liquid chromatography.

PubMed

Jandera, Pavel; Hájek, Tomás; Cesla, Petr

2010-06-01

Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions. In the second dimension, gradients are limited to a short-time period available for separation. Various types of second-dimension gradients in comprehensive LC x LC are compared: (i) "full in fraction", (ii) "segment in fraction" and (iii) "continuously shifting" gradients, applied in orthogonal LC x LC separations of phenolic acids and flavones on a polyethylene glycol column in the first dimension and two types of porous shell fused-core C18 columns in the second dimension (Ascentis Express and Kinetex). The porous shell columns provide narrow bandwidths and fast second-dimension separations at moderate operating pressure that allows important savings of the overall separation time in comprehensive LC x LC separations. The effects of the gradient type on the bandwidths, theoretical peak capacity, separation time and column pressure in the second dimension were investigated. The type of gradient program controls the range of lipophilicity of sample compounds that can be separated in the second-dimension reversed-phase time period. This range can be calibrated using alkylbenzene standards, to design the separation conditions for complete sample separation, avoiding harmful wrap around of non-eluted compounds to the subsequent second-dimension fractions.

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC-ESI-MS/MS.

PubMed

Park, Ah Yeon; Park, So-Young; Lee, Jaehyun; Jung, Mihye; Kim, Jinwoong; Kang, Sam Sik; Youm, Jeong-Rok; Han, Sang Beom

2009-10-01

Rapid, simple and reliable HPLC/UV and LC-ESI-MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C(30) column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC-ESI-MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC-ESI-MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC-ESI-MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC-ESI-MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright (c) 2009 John Wiley & Sons, Ltd.

Capillary flow-driven microfluidic device with wettability gradient and sedimentation effects for blood plasma separation.

PubMed

Maria, M Sneha; Rakesh, P E; Chandra, T S; Sen, A K

2017-03-03

We report a capillary flow-driven microfluidic device for blood-plasma separation that comprises a cylindrical well between a pair of bottom and top channels. Exposure of the well to oxygen-plasma creates wettability gradient on its inner surface with its ends hydrophilic and middle portion hydrophobic. Due to capillary action, sample blood self-infuses into bottom channel and rises up the well. Separation of plasma occurs at the hydrophobic patch due to formation of a 'self-built-in filter' and sedimentation. Capillary velocity is predicted using a model and validated using experimental data. Sedimentation of RBCs is explained using modified Steinour's model and correlation between settling velocity and liquid concentration is found. Variation of contact angle on inner surface of the well is characterized and effects of well diameter and height and dilution ratio on plasma separation rate are investigated. With a well of 1.0 mm diameter and 4.0 mm height, 2.0 μl of plasma was obtained (from <10 μl whole blood) in 15 min with a purification efficiency of 99.9%. Detection of glucose was demonstrated with the plasma obtained. Wetting property of channels was maintained by storing in DI water under vacuum and performance of the device was found to be unaffected over three weeks.

Capillary flow-driven microfluidic device with wettability gradient and sedimentation effects for blood plasma separation

PubMed Central

Maria, M. Sneha; Rakesh, P. E.; Chandra, T. S.; Sen, A. K.

2017-01-01

We report a capillary flow-driven microfluidic device for blood-plasma separation that comprises a cylindrical well between a pair of bottom and top channels. Exposure of the well to oxygen-plasma creates wettability gradient on its inner surface with its ends hydrophilic and middle portion hydrophobic. Due to capillary action, sample blood self-infuses into bottom channel and rises up the well. Separation of plasma occurs at the hydrophobic patch due to formation of a ‘self-built-in filter’ and sedimentation. Capillary velocity is predicted using a model and validated using experimental data. Sedimentation of RBCs is explained using modified Steinour’s model and correlation between settling velocity and liquid concentration is found. Variation of contact angle on inner surface of the well is characterized and effects of well diameter and height and dilution ratio on plasma separation rate are investigated. With a well of 1.0 mm diameter and 4.0 mm height, 2.0 μl of plasma was obtained (from <10 μl whole blood) in 15 min with a purification efficiency of 99.9%. Detection of glucose was demonstrated with the plasma obtained. Wetting property of channels was maintained by storing in DI water under vacuum and performance of the device was found to be unaffected over three weeks. PMID:28256564

A general strategy for performing temperature-programming in high performance liquid chromatography--further improvements in the accuracy of retention time predictions of segmented temperature gradients.

PubMed

Wiese, Steffen; Teutenberg, Thorsten; Schmidt, Torsten C

2012-01-27

In the present work it is shown that the linear elution strength (LES) model which was adapted from temperature-programming gas chromatography (GC) can also be employed for systematic method development in high-temperature liquid chromatography (HT-HPLC). The ability to predict isothermal retention times based on temperature-gradient as well as isothermal input data was investigated. For a small temperature interval of ΔT=40°C, both approaches result in very similar predictions. Average relative errors of predicted retention times of 2.7% and 1.9% were observed for simulations based on isothermal and temperature-gradient measurements, respectively. Concurrently, it was investigated whether the accuracy of retention time predictions of segmented temperature gradients can be further improved by temperature dependent calculation of the parameter S(T) of the LES relationship. It was found that the accuracy of retention time predictions of multi-step temperature gradients can be improved to around 1.5%, if S(T) was also calculated temperature dependent. The adjusted experimental design making use of four temperature-gradient measurements was applied for systematic method development of selected food additives by high-temperature liquid chromatography. Method development was performed within a temperature interval from 40°C to 180°C using water as mobile phase. Two separation methods were established where selected food additives were baseline separated. In addition, a good agreement between simulation and experiment was observed, because an average relative error of predicted retention times of complex segmented temperature gradients less than 5% was observed. Finally, a schedule of recommendations to assist the practitioner during systematic method development in high-temperature liquid chromatography was established. Copyright © 2011 Elsevier B.V. All rights reserved.

Selected retinoids: determination by isocratic normal-phase HPLC.

PubMed

Klvanova, J; Brtko, J

2002-09-01

Retinol (ROL), retinal (RAL) and retinoic acid (RA) are physiologically active forms of vitamin A. All-trans retinoic acid (ATRA) can be formed by oxidation from all-trans retinal (ATRAL). Isomerization of RA is considered to be an important metabolic pathway of retinoids. RA isomers transactivate various response pathways via their cognate nuclear receptors that act as ligand inducible transcription factors. The aim of this study was to establish a rapid and simple method for determination of ATRA, 13-cis retinoic acid (13CRA) and ATRAL by HPLC. In our laboratory, we slightly modified the method of Miyagi et al. (2001) and separated ATRA, 13CRA and ATRAL by simple isocratic normal phase HPLC. Both retinoic acid isomers and ATRAL were eluted within 13 min and all components were well resolved. The coefficients of variation (C.V.) for RAs and RAL were from 3.0 to 5.4 %.

Comparison of the effects of Mylabris and Acanthopanax senticosus on promising cancer marker polyamines in plasma of a Hepatoma-22 mouse model using HPLC-ESI-MS.

PubMed

Wang, Qian; Wang, Yixiang; Liu, Ran; Yan, Xu; Li, Yujiao; Fu, Hui; Bi, Kaishun; Li, Qing

2013-02-01

A simple and sensitive method for the simultaneous determination of plasma concentrations of five polyamines in normal and Hepatoma-22 mice, and mice treated with Mylabris and Acanthopanax senticosus was developed by HPLC-ESI-MS. Male Kunming mice were divided into nine groups, a control group (inoculation without treatment), a positive group (Cyclophosphamide), treatment groups [Mylabris (4, 8, 16 mg/kg), Acanthopanax senticosus (6, 12, 24 g/kg)] and a normal group (without inoculation). Twenty-four hours after the last administration, plasma samples were collected. The derived polyamines were separated on a C(18) column by a gradient elution using methanol-water with excellent linearity within the range from 2.5 to 1000 ng/mL. Polyamines were confirmed as useful biochemical markers of hepatoma. The differences in anti-cancer therapeutic efficacy between Mylabris and Acanthopanax senticosus might contribute to the variability of polyamine levels in vivo. This HPLC-ESI-MS method was successfully applied to investigate the relationship between polyamines and cancer in mice and might be a useful method to test the activity of potential anti-tumor drugs. Copyright © 2012 John Wiley & Sons, Ltd.

Surface confined ionic liquid as a stationary phase for HPLC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Qian; Baker, Gary A; Baker, Sheila N

Trimethoxysilane ionosilane derivatives of room temperature ionic liquids based on alkylimidazolium bromides were synthesized for attachment to silica support material. The derivatives 1-methyl-3-(trimethoxysilylpropyl)imidazolium bromide and 1-butyl-3-(trimethoxysilylpropyl)imidazolium bromide were used to modify the surface of 3 {micro}m diameter silica particles to act as the stationary phase for HPLC. The modified particles were characterized by thermogravimetric analysis (TGA) and {sup 13}C and {sup 29}Si NMR spectroscopies. The surface modification procedure rendered particles with a surface coverage of 0.84 {micro}mol m{sup -2} for the alkylimidazolium bromide. The ionic liquid moiety was predominantly attached to the silica surface through two siloxane bonds of themore » ionosilane derivative (63%). Columns packed with the modified silica material were tested under HPLC conditions. Preliminary evaluation of the stationary phase for HPLC was performed using aromatic carboxylic acids as model compounds. The separation mechanism appears to involve multiple interactions including ion exchange, hydrophobic interaction, and other electrostatic interactions.« less

Recursion equations in predicting band width under gradient elution.

PubMed

Liang, Heng; Liu, Ying

2004-06-18

The evolution of solute zone under gradient elution is a typical problem of non-linear continuity equation since the local diffusion coefficient and local migration velocity of the mass cells of solute zones are the functions of position and time due to space- and time-variable mobile phase composition. In this paper, based on the mesoscopic approaches (Lagrangian description, the continuity theory and the local equilibrium assumption), the evolution of solute zones in space- and time-dependent fields is described by the iterative addition of local probability density of the mass cells of solute zones. Furthermore, on macroscopic levels, the recursion equations have been proposed to simulate zone migration and spreading in reversed-phase high-performance liquid chromatography (RP-HPLC) through directly relating local retention factor and local diffusion coefficient to local mobile phase concentration. This new approach differs entirely from the traditional theories on plate concept with Eulerian description, since band width recursion equation is actually the accumulation of local diffusion coefficients of solute zones to discrete-time slices. Recursion equations and literature equations were used in dealing with same experimental data in RP-HPLC, and the comparison results show that the recursion equations can accurately predict band width under gradient elution.

Combined HPLC-CUPRAC (cupric ion reducing antioxidant capacity) assay of parsley, celery leaves, and nettle.

PubMed

Yildiz, Leyla; Başkan, Kevser Sözgen; Tütem, Esma; Apak, Reşat

2008-10-19

This study aims to identify the essential antioxidant compounds present in parsley (Petroselinum sativum) and celery (Apium graveolens) leaves belonging to the Umbelliferae (Apiaceae) family, and in stinging nettle (Urtica dioica) belonging to Urticaceae family, to measure the total antioxidant capacity (TAC) of these compounds with CUPRAC (cupric ion reducing antioxidant capacity) and ABTS spectrophotometric methods, and to correlate the TAC with high performance liquid chromatography (HPLC) findings. The CUPRAC spectrophotometric method of TAC assay using copper(II)-neocuproine (2,9-dimethyl-1,10-phenanthroline) as the chromogenic oxidant was developed in our laboratories. The individual antioxidant constituents of plant extracts were identified and quantified by HPLC on a C18 column using a modified mobile phase of gradient elution comprised of MeOH-0.2% o-phosphoric acid and UV detection for polyphenols at 280 nm. The TAC values of HPLC-quantified antioxidant constituents were found, and compared for the first time with those found by CUPRAC. The TAC of HPLC-quantified compounds accounted for a relatively high percentage of the observed CUPRAC capacities of plant extracts, namely 81% of nettle, 60-77% of parsley (in different hydrolyzates of extract and solid sample), and 41-57% of celery leaves (in different hydrolyzates). The CUPRAC total capacities of the 70% MeOH extracts of studied plants (in the units of mmol trolox g(-1)plant) were in the order: celery leaves>nettle>parsley. The TAC calculated with the aid of HPLC-spectrophotometry did not compensate for 100% of the CUPRAC total capacities, because all flavonoid glycosides subjected to hydrolysis were either not detectable with HPLC, or not converted to the corresponding aglycons (i.e., easily detectable and quantifiable with HPLC) during the hydrolysis step.

Validation of a simple HPLC-UV method for rifampicin determination in plasma: Application to the study of rifampicin arteriovenous concentration gradient.

PubMed

Goutal, Sébastien; Auvity, Sylvain; Legrand, Tiphaine; Hauquier, Fanny; Cisternino, Salvatore; Chapy, Hélène; Saba, Wadad; Tournier, Nicolas

2016-05-10

In clinical practice, rifampicin exposure is estimated from its concentration in venous blood samples. In this study, we hypothesized that differences in rifampicin concentration may exist between arterial and venous plasma. An HPLC-UV method for determining rifampicin concentration in plasma using rifapentine as an internal standard was validated. The method, which requires a simple protein precipitation procedure as sample preparation, was performed to compare venous and arterial plasma kinetics after a single therapeutic dose of rifampicin (8.6 mg/kg i.v, infused over 30 min) in baboons (n=3). The method was linear from 0.1 to 40 μg mL(-1) and all validation parameters fulfilled the international requirements. In baboons, rifampicin concentration in arterial plasma was higher than in venous plasma. Arterial Cmax was 2.1±0.2 fold higher than venous Cmax. The area under the curve (AUC) from 0 to 120 min was ∼80% higher in arterial plasma, indicating a significant arteriovenous concentration gradient in early rifampicin pharmacokinetics. Arterial and venous plasma concentrations obtained 6h after rifampicin injection were not different. An important arteriovenous equilibration delay for rifampicin pharmacokinetics is reported. Determination in venous plasma concentrations may considerably underestimate rifampicin exposure to organs during the distribution phase. Copyright © 2016 Elsevier B.V. All rights reserved.

New validated method for piracetam HPLC determination in human plasma.

PubMed

Curticapean, Augustin; Imre, Silvia

2007-01-10

The new method for HPLC determination of piracetam in human plasma was developed and validated by a new approach. The simple determination by UV detection was performed on supernatant, obtained from plasma, after proteins precipitation with perchloric acid. The chromatographic separation of piracetam under a gradient elution was achieved at room temperature with a RP-18 LiChroSpher 100 column and aqueous mobile phase containing acetonitrile and methanol. The quantitative determination of piracetam was performed at 200 nm with a lower limit of quantification LLQ=2 microg/ml. For this limit, the calculated values of the coefficient of variation and difference between mean and the nominal concentration are CV%=9.7 and bias%=0.9 for the intra-day assay, and CV%=19.1 and bias%=-7.45 for the between-days assay. For precision, the range was CV%=1.8/11.6 in the intra-day and between-days assay, and for accuracy, the range was bias%=2.3/14.9 in the intra-day and between-days assay. In addition, the stability of piracetam in different conditions was verified. Piracetam proved to be stable in plasma during 4 weeks at -20 degrees C and for 36 h at 20 degrees C in the supernatant after protein precipitation. The new proposed method was used for a bioequivalence study of two medicines containing 800 mg piracetam.

Separation and quantification of 15 carotenoids by reversed phase high performance liquid chromatography coupled to diode array detection with isosbestic wavelength approach.

PubMed

Mitrowska, Kamila; Vincent, Ursula; von Holst, Christoph

2012-04-13

The manuscript presents the development of a new reverse phase high performance liquid chromatography (RP-HPLC) photo diode array detection method allowing the separation and quantification of 15 carotenoids (adonirubin, adonixanthin, astaxanthin, astaxanthin dimethyl disuccinate, asteroidenone, beta-apo-8'-carotenal, beta-apo-8'-carotenoic acid ethyl ester, beta-carotene, canthaxanthin, capsanthin, citranaxanthin, echinenone, lutein, lycopene, and zeaxanthin), 10 of which are feed additives authorised within the European Union. The developed method allows for the reliable determination of the total carotenoid content in one run using the corresponding E-isomer as calibration standard while taking into account the E/Z-isomers composition. This is a key criterion for the application of the method, since for most of the analytes included in this study analytical standards are only available for the E-isomers. This goal was achieved by applying the isosbestic concept, in order to identify specific wavelengths, at which the absorption coefficients are identical for all stereoisomers concerned. The second target referred to the optimisation of the LC conditions. By means of an experimental design, an optimised RP-HPLC method was developed allowing for a sufficient chromatographic separation of all carotenoids. The selected method uses a Suplex pKb-100 HPLC column and applying a gradient with a mixture of acetonitrile, tert-butyl-methyl ether and water as mobile phases. The limits of detection and limits of quantification ranged from 0.06 mg L(-1) to 0.14 mg L(-1) and from 0.20 mg L(-1) to 0.48 mg L(-1), respectively. Copyright © 2012 Elsevier B.V. All rights reserved.

[Determination of benzyl glucosinolate in Lepidium meyenii from different regions by HPLC].

PubMed

Tang, Lin; Yin, Hong-jun; Si, Cong-cong; Hu, Xiao-yan; Long, Zheng-hai

2015-12-01

The content of benzyl isothiocyanate (BITC) which as the enzymatic hydrolysis product of benzyl glucosinolate through thioglucosidase was determined by HPLC. The content of benzyl isothiocyanate (BITC) which as the enzymatic hydrolysis product of benzyl glucosinolate through thioglucosidase was determined by HPLC. The chromatography condition was as follows: Kaseisorb LC ODS 2000 (4.6 mm x 150 mm, 5 min) column with the mobile phase of acetonitrile(A)-water( B) under gradient elution (0-5 min, 3%-8% A; 5-9 min, 8%-48% A; 9-23 min, 48%-62% A; 23-28 min, 62%-99% A); the flow rate was 1.0 mL x min(-1) with 10 microL injection volume; detection wavelength was 246 nm and temperature of column was 40 degrees C. The content of benzyl glucosinolate was in the range of 10.76-17.91 g x L(-1). The method is simple, accurate and good reproducibility which can be used for the determination of benzyl glucosinolate in Lepidium meyenii, effectively.

[Study on HPLC fingerprint of 11 Taraxacum species in northeast of China].

PubMed

Zhu, Dan; Zhao, Xin; Xu, Qiao; Ning, Wei

2011-04-01

To study the RP-HPLC fingerprints of 11 plants in the genus Taraxacum for their quality control. The fingerprints were determined using an Agilent 1100 series instrument system. Chromatographic analyses were performed on a Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) analytical column,eluted with methanol and water containing 0.5% acetic acid as the mobile phases in gradient elution at the flow rate of 1.0 mL x min(-1). The detection wavelength was 323 nm. The temperature of column was 35 degrees C. Eleven species of Taraxacum in northeast of China were detected respectively. Twenty-five common peaks existed in 11 RP-HPLC fingerprints. By comparing the retention time and the on-line UV spectra, peaks No. 10, No. 12, No. 16 and No. 25 were identified as chlorogenic acid, caffeic acid, p-coumaroy acid and luteolin respectively. The analytical method with good precision and reproducibility can be useful in the quality control of Taraxacum plants.

Magnetic separation of algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nath, Pulak; Twary, Scott N.

Described herein are methods and systems for harvesting, collecting, separating and/or dewatering algae using iron based salts combined with a magnetic field gradient to separate algae from an aqueous solution.

Simultaneous Speciation of Arsenic, Selenium, and Chromium by HPLC-ICP-MS

USGS Publications Warehouse

Wolf, Ruth E.; Morman, Suzette A.; Morrison, Jean M.; Lamothe, Paul J.

2008-01-01

An adaptation of an analytical method developed for chromium speciation has been utilized for the simultaneous determination of As(III), As(V), Se(IV), Se(VI), Cr(III), and Cr(VI) species using high performance liquid chromatography (HPLC) separation with ICP-MS detection. Reduction of interferences for the determination of As, Se, and Cr by ICP-MS is a major consideration for this method. Toward this end, a Dynamic Reaction Cell (DRC) ICP-MS system was used to detect the species eluted from the chromatographic column. A variety of reaction cell gases and conditions may be utilized, and the advantages and limitations of the gases tested to date will be presented and discussed. The separation and detection of the As, Se, and Cr species of interest can be achieved using the same chromatographic conditions in less than 2 minutes by complexing the Cr(III) with EDTA prior to injection on the HPLC column. Practical aspects of simultaneous speciation analysis will be presented and discussed, including issues with HPLC sample vial contamination, standard and sample contamination, species stability, and considerations regarding sample collection and preservation methods. The results of testing to determine the method's robustness to common concomitant element and anion effects will also be discussed. Finally, results will be presented using the method for the analysis of a variety of environmental and geological samples including waters, soil leachates and simulated bio-fluid leachates.

Simultaneous HPLC quantitative analysis of active compounds in leaves of Moringa oleifera Lam.

PubMed

Vongsak, Boonyadist; Sithisarn, Pongtip; Gritsanapan, Wandee

2014-08-01

Moringa oleifera Lam. has been used as a traditional medicine for the treatment of numerous diseases. A simultaneous high-performance liquid chromatography (HPLC) analysis was developed and validated for the determination of the contents of crypto-chlorogenic acid, isoquercetin and astragalin, the primary antioxidative compounds, in M. oleifera leaves. HPLC analysis was successfully conducted by using a Hypersil BDS C18 column, eluted with a gradient of methanol-1% acetic acid with a flow rate of 1 mL/min, and detected at 334 nm. Parameters for the validation included linearity, precision, accuracy and limits of detection and quantitation. The developed HPLC method was precise, with relative standard deviation < 2%. The recovery values of crypto-chlorogenic acid, isoquercetin and astragalin in M. oleifera leaf extracts were 98.50, 98.47 and 98.59%, respectively. The average contents of these compounds in the dried ethanolic extracts of the leaves of M. oleifera collected from different regions of Thailand were 0.081, 0.120 and 0.153% (w/w), respectively. The developed HPLC method was appropriate and practical for the simultaneous analysis of crypto-chlorogenic acid, isoquercetin and astragalin in the leaf extract of M. oleifera. This work is valuable as guidance for the standardization of the leaf extracts and pharmaceutical products of M. oleifera. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Simultaneous quantitation of chloroquine and primaquine by UPLC-DAD and comparison with a HPLC-DAD method.

PubMed

Miranda, Tiago A; Silva, Pedro H R; Pianetti, Gerson A; César, Isabela C

2015-01-28

Chloroquine and primaquine are the first-line treatment recommended by World Health Organization for malaria caused by Plasmodium vivax. Since the problem of counterfeit or substandard anti-malarials is well established all over the world, the development of rapid and reliable methods for quality control analysis of these drugs is essential. Thus, the aim of this study was to develop and validate a novel UPLC-DAD method for simultaneously quantifying chloroquine and primaquine in tablet formulations. The UPLC separation was carried out using a Hypersil C18 column (50 × 2.1 mm id; 1.9 μm particle size) and a mobile phase composed of acetonitrile (A) and 0.1% aqueous triethylamine, pH 3.0 adjusted with phosphoric acid (B), at a flow rate 0.6 mL/min. Gradient elution was employed. UV detection was performed at 260 nm. UPLC method was fully validated and the results were compared to a conventional HPLC-DAD method for the analysis of chloroquine and primaquine in tablet formulations. UPLC method was shown to be linear (r2 > 0.99), precise (CV < 2.0%), accurate (recovery rates from 98.11 to 99.83%), specific, and robust. No significant differences were observed between the chloroquine and primaquine contents obtained by UPLC and HPLC methods. However, UPLC method promoted faster analyses, better chromatographic performance and lower solvent consumption. The developed UPLC method was shown to be a rapid and suitable technique to quantify chloroquine and primaquine in pharmaceutical preparations and may be successfully employed for quality control analysis.

Detection of monohydroxylated polycyclic aromatic hydrocarbons in urine and particulate matter using LC separations coupled with integrated SPE and fluorescence detection or coupled with high-resolution time-of-flight mass spectrometry.

PubMed

Lintelmann, Jutta; Wu, Xiao; Kuhn, Evelyn; Ritter, Sebastian; Schmidt, Claudia; Zimmermann, Ralf

2018-05-01

A high-performance liquid chromatographic (HPLC) method with integrated solid-phase extraction for the determination of 1-hydroxypyrene and 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene in urine was developed and validated. After enzymatic treatment and centrifugation of 500 μL urine, 100 μL of the sample was directly injected into the HPLC system. Integrated solid-phase extraction was performed on a selective, copper phthalocyanine modified packing material. Subsequent chromatographic separation was achieved on a pentafluorophenyl core-shell column using a methanol gradient. For quantification, time-programmed fluorescence detection was used. Matrix-dependent recoveries were between 94.8 and 102.4%, repeatability and reproducibility ranged from 2.2 to 17.9% and detection limits lay between 2.6 and 13.6 ng/L urine. A set of 16 samples from normally exposed adults was analyzed using this HPLC-fluorescence detection method. Results were comparable with those reported in other studies. The chromatographic separation of the method was transferred to an ultra-high-performance liquid chromatography pentafluorophenyl core-shell column and coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF-MS). The resulting method was used to demonstrate the applicability of LC-HR-TOF-MS for simultaneous target and suspect screening of monohydroxylated polycyclic aromatic hydrocarbons in extracts of urine and particulate matter. Copyright © 2018 John Wiley & Sons, Ltd.

Determination of phenolic compounds derived from hydrolysable tannins in biological matrices by RP-HPLC.

PubMed

Díez, María Teresa; García del Moral, Pilar; Resines, José Antonio; Arín, María Jesús

2008-08-01

An RP-HPLC method for the determination of four phenolic compounds: gallic acid (GA), pyrogallol (PY), resorcinol (RE) and ellagic acid (EA), derived from hydrolysable tannins is reported. Separation was achieved on a SunFire C18 (250 x 4.6 mm id, 5 microm) column at 40 degrees C with gradient elution. UV detection at 280 nm was applied. The developed method was validated in terms of linearity, accuracy and precision. Satisfactory repeatability and between day precision were noticed with RSD values lower than 3%. Recoveries from different biological samples ranged from 91.50 to 105.25%. The LODs were estimated as 1.70 mg/L for PY, 1.68 mg/L for GA, 1.52 mg/L for RE and 0.98 mg/L for EA with a 20 microL injection volume. The method was applied for the determination of these compounds in oak leaves and in ruminal fluid and urine samples taken from beef cattle fed with oak leaves. The proposed method could be used in ruminant nutrition studies to verify the effect that a diet rich in tannins have on ruminal fermentation and to determine the toxicity of these compounds.

Contribution of PCR Denaturing Gradient Gel Electrophoresis Combined with Mixed Chromatogram Software Separation for Complex Urinary Sample Analysis.

PubMed

Kotásková, Iva; Mališová, Barbora; Obručová, Hana; Holá, Veronika; Peroutková, Tereza; Růžička, Filip; Freiberger, Tomáš

2017-01-01

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment. © 2017 S. Karger AG, Basel.

[Simultaneous analysis of four diuretic drugs by HPLC and its application to health food supplements advertising weight reduction].

PubMed

Goto, Tomomi; Mikami, Eiichi; Ohno, Tsutomu; Matsumoto, Hiroshi

2002-04-01

A high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of triamterene, trichlormethiazide, furosemide and spironolactone is presented for application in the examination of health food supplements advertising weight reduction and in the analysis of pharmaceuticals. The HPLC assay was performed under gradient conditions using a Wakosil ODS 5C18 column (5 microns, 150 x 4.6 mm i.d.). The mobile phase consisted of a gradient program with a mixture of water and acetonitrile containing 0.1% triethylamine adjusted with phosphoric acid to pH 3.0: from 0 to 6 min, 15% acetonitrile; from 6 to 20 min, linear gradient from 15 to 50% acetonitrile; and from 20 to 40 min, 50% acetonitrile. The column effluent was monitored from 0 to 20 min at 260 nm and from 20 to 40 min at 235 nm. The calibration curves of the four drugs showed good linearity and the correlation coefficients were better than 0.999 in all cases. The lower limits of detection were approximately 40 ng for each drug. Commercially available health food supplements and pharmaceuticals were analyzed after extraction with a mixture of methanol and acetic acid (99:1). The procedure described here is suitable for the screening of four diuretic drugs in adulterated supplements and for the quality control of pharmaceuticals with minimal sample preparation.

Molecular characterization and bio-functional property determination using SDS-PAGE and RP-HPLC of protein fractions from two Nigella species.

PubMed

Alu'datt, Muhammad H; Rababah, Taha; Alhamad, Mohammad N; Alodat, Moh'd; Al-Mahasneh, Majdi A; Gammoh, Sana; Ereifej, Khalil; Almajwal, Ali; Kubow, Stan

2017-09-01

This study aimed to investigate the molecular and bio-functional properties of protein fractions from Nigella damascena and Nigella arvensis, including the albumin, globulin, glutein-1, glutein-2 and prolamin fractions. Protein subunits were not observed in globulin and prolamin fractions. No peaks appeared in RP-HPLC chromatograms of globulin for either species. Two predominant peaks were observed in the RP-HPLC profiles of all protein fractions. Proteins separated by RP-HPLC have potential inhibitory and antioxidant activities in all fractions. Optimum ACE-inhibitory and antioxidant activities of proteins separated by RP-HPLC were observed in glutein-2 and albumin, respectively, for both species. For pepsin and combined pepsin-trypsin hydrolyses, the highest degree of hydrolysis (DH) was obtained in glutein-2 fraction of Nigella arvensis. Highest ACE-inhibitory activity of hydrolyzed protein fractions was found at 4h via pepsin hydrolysis in globulin fraction of Nigella damascena. Highest antioxidant activities of hydrolyzed protein fractions were found in glutelin-2 for both species. Copyright © 2017 Elsevier Ltd. All rights reserved.

Monolithic stationary phases with a longitudinal gradient of porosity.

PubMed

Urban, Jiří; Hájek, Tomáš; Svec, Frantisek

2017-04-01

The duration of the hypercrosslinking reaction has been used to control the extent of small pores formation in polymer-based monolithic stationary phases. Segments of five columns hypercrosslinked for 30-360 min were coupled via zero-volume unions to prepare columns with segmented porosity gradients. The steepness of the porosity gradient affected column efficiency, mass transfer resistance, and separation of both small-molecule alkylbenzenes and high-molar-mass polystyrene standards. In addition, the segmented column with the steepest porosity gradient was prepared as a single column with a continuous porosity gradient. The steepness of porosity gradient in this type column was tuned. Compared to a completely hypercrosslinked column, the column with the shallower gradient produced comparable size-exclusion separation of polystyrene standards but allowed higher column permeability. The completely hypercrosslinked column and the column with porosity gradient were successfully coupled in online two-dimensional liquid chromatography of polymers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An Experimental Design Approach for Impurity Profiling of Valacyclovir-Related Products by RP-HPLC

PubMed Central

Katakam, Prakash; Dey, Baishakhi; Hwisa, Nagiat T; Assaleh, Fathi H; Chandu, Babu R; Singla, Rajeev K; Mitra, Analava

2014-01-01

Abstract Impurity profiling has become an important phase of pharmaceutical research where both spectroscopic and chromatographic methods find applications. The analytical methodology needs to be very sensitive, specific, and precise which will separate and determine the impurity of interest at the 0.1% level. Current research reports a validated RP-HPLC method to detect and separate valacyclovir-related impurities (Imp-E and Imp-G) using the Box-Behnken design approach of response surface methodology. A gradient mobile phase (buffer: acetonitrile as mobile phase A and acetonitrile: methanol as mobile phase B) was used. Linearity was found in the concentration range of 50–150 μg/mL. The mean recovery of impurities was 99.9% and 103.2%, respectively. The %RSD for the peak areas of Imp-E and Imp-G were 0.9 and 0.1, respectively. No blank interferences at the retention times of the impurities suggest the specificity of the method. The LOD values were 0.0024 μg/mL for Imp-E and 0.04 μg/mL for Imp-G and the LOQ values were obtained as 0.0082 μg/mL and 0.136 μg/mL, respectively, for the impurities. The S/N ratios in both cases were within the specification limits. Proper peak shapes and satisfactory resolution with good retention times suggested the suitability of the method for impurity profiling of valacyclovir-related drug substances. PMID:25853072

Comprehensive two-dimensional chromatography with coupling of reversed phase high performance liquid chromatography and supercritical fluid chromatography.

PubMed

Stevenson, Paul G; Tarafder, Abhijit; Guiochon, Georges

2012-01-13

A 2D comprehensive chromatographic separation of blackberry sage fragrant oil was performed by using HPLC in the first dimension and SFC in the second. A C(18)-bonded silica column eluted with an ACN gradient was used in the HPLC dimension and an amino-bonded silica column eluted with ACN as a modifier in the SFC dimension. This 2D separation was completed in the off-line mode, the fractions from the HPLC column being collected and injected in the SFC column. The retention factors on the two columns have a -0.757 correlation coefficient. The method provides a practical peak capacity of 2400 in 280 min. The first eluted peaks in HPLC are the last ones eluted in SFC and vice versa. The results demonstrate that the coupling of an HPLC and an SFC separation have a great potential for 2D chromatographic separations. Copyright © 2011 Elsevier B.V. All rights reserved.

On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.).

PubMed

Bandoniene, Donata; Murkovic, Michael

2002-04-24

An on-line HPLC-DPPH screening method for phenolic antioxidants in apple methanol/water (80:20, v/v) extracts was applied. The determination of antioxidants was based on a decrease in absorbance at 515 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-diphenyl-1-picrylhydrazyl radicals (DPPH*). Each of the antioxidants separated by the HPLC column was observed as a negative peak corresponding to its antioxidative activity. The on-line method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area on concentration of the reference antioxidants was observed. For validation of the on-line method the limit of detection, LOD (microg/mL), and the limit of quantification, LOQ (microg/mL), of the phenolic compounds were determined. Comparison of the UV and DPPH radical quenching chromatograms with authentic compounds identified catechin, chlorogenic acid, caffeic acid, epicatechin, and phloridzin in the apple cultivars (Lobo, Golden Delicious, and Boskoop), and the distribution of total antioxidant activity was calculated.

HPLC-ELSD Quantification and Centrifugal Partition Chromatography Isolation of 8-O-Acetylharpagide from Oxera coronata (Lamiaceae).

PubMed

Remeur, Camille; Le Borgne, Erell; Gauthier, Léa; Grougnet, Raphaël; Deguin, Brigitte; Poullain, Cyril; Litaudon, Marc

2017-05-01

Iridoid glycosides possess highly functionalised monoterpenoid aglycon with several contiguous stereocentres. For the most common, they are often present in quantities reaching several percentage of the fresh plant weight, and thus they may be regarded as starting material for the synthesis of a number of new chiral and bioactive molecules. To quantify and to isolate 8-O-acetylharpagide (AH) from several extracts of Oxera coronata R.P.J. de Kok, a Lamiaceae species endemic to New Caledonia, using HPLC-ELSD (evaporative light scattering detector) and centrifugal partition chromatography (CPC). Oxera coronata produces high amounts of AH in leaves, twigs and fruits. Water and methanol extracts of these plant parts were prepared. The content of AH in each extract was quantified by HPLC-ELSD, using acetonitrile-water (+0.1% formic acid) gradient elution. The HPLC method was validated for precision, linearity, limit of detection (LOD), limit of quantification (LOQ) and accuracy. A ternary solvent system ethyl acetate/n-propanol/water (3:2:5, v/v/v) was selected and applied to recover the target compound using Spot CPC from the leaves aqueous extract. HPLC-ELSD analysis followed by CPC purification led to the efficient isolation of AH from O. coronata leaves aqueous extract. HPLC-ELSD has proven to be a well-adapted detection and quantification method for iridoid glycosides, while CPC confirmed to be an efficient technique for the isolation of polar compounds. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous determination of 12 chemical constituents in the traditional Chinese Medicinal Prescription Xiao-Yao-San-Jia-Wei by HPLC coupled with photodiode array detection.

PubMed

Zhang, Hongmin; Chen, Shiwei; Qin, Feng; Huang, Xi; Ren, Ping; Gu, Xinqi

2008-12-15

An HPLC-photodiode array (PDA) detection method was established for the simultaneous determination of 12 components in Xiao-Yao-San-Jia-Wei (XYSJW): geniposide, puerarin, paeoniflorin, ferulic acid, liquiritin, hesperidin, naringin, paeonol, daidzein, glycyrrhizic acid, honokiol, and magnolol. These were separated in less than 70 min using a Waters Symmetry Shield RP 18 column with gradient elution using (A) acetonitrile, (B) water, and (C) acetic acid at a flow rate of 1 ml/min, and with a PDA detector. All calibration curves showed good linear regression (r(2)>0.9992) within the test ranges. The method was validated for specificity, accuracy, precision, and limits of detection. The proposed method enables in a single run the simultaneous identification and determination for quality control of 12 multi-structural components of XYSJW forming the basis of its therapeutic effect.

New solvent systems for gradient counter-current chromatography in separation of betanin and its derivatives from processed Beta vulgaris L. juice.

PubMed

Spórna-Kucab, Aneta; Garrard, Ian; Ignatova, Svetlana; Wybraniec, Sławomir

2015-02-06

Betalains, natural plant pigments, are beneficial compounds due to their antioxidant and possible chemoprotective properties. A mixture of betalains: betanin/isobetanin, decarboxybetanins and neobetanin from processed red beet roots (Beta vulgaris L.) juice was separated in food-grade, gradient solvent systems using high-performance counter-current chromatography (HPCCC). The decarboxylated and dehydrogenated betanins were obtained by thermal degradation of betanin/isobetanin from processed B. vulgaris L. juice under mild conditions. Two solvent systems (differing in their composition by phosphoric acid and ethanol volume gradient) consisting of BuOH-EtOH-NaClsolution-H2O-H3PO4 (v/v/v/v/v, 1300:200-1000:1300:700:2.5-10) in the 'tail-to-head' mode were run. The flow rate of the mobile phase (organic phase) was 1.0 or 2.0 ml/min and the column rotation speed was 1,600 rpm (20°C). The retention of the solvent system stationary phase (aqueous phase) was ca. 80%. The system with the acid and ethanol volume gradient consisting of BuOH-EtOH-NaClsolution-H2O-H3PO4 (v/v/v/v/v, 1300:200-240:1300:700:2.5-4.5) pumped at 2.0 ml/min was the most effective for a separation of betanin/isobetanin, 17-decarboxy-betanin/-isobetanin, 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin pairs as well as neobetanin. The pigments were detected by LC-DAD and LC-MS. The results are crucial in the application of completely food-grade solvent systems in separation of food-grade compounds as well, and the systems can possibly be extended to other ionizable and polar compounds with potential health benefits. In particular, the method is applicable for the isolation and purification of betalains present in such rich sources as B. vulgaris L. roots as well as cacti fruits and Amaranthaceae flowering plants due to modification possibilities of the solvent systems polarity. Copyright © 2014 Elsevier B.V. All rights reserved.

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases.

PubMed

Tsonev, Latchezar I; Hirsh, Allen G

2008-07-25

pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt.

Authentication and Quantitation of Fraud in Extra Virgin Olive Oils Based on HPLC-UV Fingerprinting and Multivariate Calibration

PubMed Central

Carranco, Núria; Farrés-Cebrián, Mireia; Saurina, Javier

2018-01-01

High performance liquid chromatography method with ultra-violet detection (HPLC-UV) fingerprinting was applied for the analysis and characterization of olive oils, and was performed using a Zorbax Eclipse XDB-C8 reversed-phase column under gradient elution, employing 0.1% formic acid aqueous solution and methanol as mobile phase. More than 130 edible oils, including monovarietal extra-virgin olive oils (EVOOs) and other vegetable oils, were analyzed. Principal component analysis results showed a noticeable discrimination between olive oils and other vegetable oils using raw HPLC-UV chromatographic profiles as data descriptors. However, selected HPLC-UV chromatographic time-window segments were necessary to achieve discrimination among monovarietal EVOOs. Partial least square (PLS) regression was employed to tackle olive oil authentication of Arbequina EVOO adulterated with Picual EVOO, a refined olive oil, and sunflower oil. Highly satisfactory results were obtained after PLS analysis, with overall errors in the quantitation of adulteration in the Arbequina EVOO (minimum 2.5% adulterant) below 2.9%. PMID:29561820

Determination of arsenic species and arsenosugars in marine samples by HPLC-ICP-MS.

PubMed

Hirata, Shizuko; Toshimitsu, Hideki

2005-10-01

Arsenic-speciation analysis in marine samples was performed by high-pressure liquid chromatography (HPLC) with ICP-MS detection. Separation of eight arsenic species--As(III), MMA, DMA, As(V), AB, TMAO, AC and TeMAs(+)--was achieved on a C(18) column with isocratic elution (pH 3.0), under which conditions As(III) and MMA co-eluted. The entire separation was accomplished in 15 min. The HPLC-ICP-MS detection limits for the eight arsenic species were in the range 0.03-0.23 microg L(-1) based on 3 sigma for the blank response (n=5). The precision was calculated to be 2.4-8.0% (RSD) for the eight species. The method was successfully applied to several marine samples, e.g. oysters, fish, shrimps, and marine algae. Low-power microwave digestion was employed for extraction of arsenic from seafood products; ultrasonic extraction was employed for the extraction of arsenic from seaweeds. Separation of arsenosugars was achieved on an anion-exchange column. Concentrations of arsenosugars 2, 3, and 4 in marine algae were in the range 0.18-9.59 microg g(-1).

HPLC-based metabolic profiling and quality control of leaves of different Panax species

PubMed Central

Yang, Seung-Ok; Lee, Sang Won; Kim, Young Ock; Sohn, Sang-Hyun; Kim, Young Chang; Hyun, Dong Yoon; Hong, Yoon Pyo; Shin, Yu Su

2013-01-01

Leaves from Panax ginseng Meyer (Korean origin and Chinese origin of Korean ginseng) and P. quinquefolius (American ginseng) were harvested in Haenam province, Korea, and were analyzed to investigate patterns in major metabolites using HPLC-based metabolic profiling. Partial least squares discriminant analysis (PLS-DA) was used to analyze the HPLC chromatogram data. There was a clear separation between Panax species and/or origins from different countries in the PLS-DA score plots. The ginsenoside compounds of Rg1, Re, Rg2, Rb2, Rb3, and Rd in Korean leaves were higher than in Chinese and American ginseng leaves, and the Rb1 level in P. quinquefolius leaves was higher than in P. ginseng (Korean origin or Chinese origin). HPLC chromatogram data coupled with multivariate statistical analysis can be used to profile the metabolite content and undertake quality control of Panax products. PMID:23717177

Phototoxicity testing by online irradiation and HPLC.

PubMed

Schröder, Sven; Surmann, J P

2006-11-01

A high-performance liquid chromatography (HPLC) system was developed for the determination of drug photostability and phototoxicity based on an automated column-switching system with aqueous online UV-A irradiation and hyphenated organic separation of the drug and its photoproducts. The photoreactor is built with an poly(ethylene-co-tetrafluoroethylene) (ETFE) reaction coil knitted around a UV-A light source. The chromatographic separation was performed with two special C18 columns, which are also suitable for using with pure water as eluent. Degradation of chlorpromazine (CPZ) by ultraviolet light was investigated at pH 7 and pH 3. Furthermore chlorpromazine was irradiated in the presence of guanosine-5-monophosphate (GMP) in pH 7 buffered solution, leading to a new photoproduct. In the pH 3 irradiation studies of CPZ and GMP, no reaction was detected between the molecules.

Ecohydrologic Separation of Plant Life Forms Across A Soil Moisture Gradient in a Montane Wetland

NASA Astrophysics Data System (ADS)

Mercer, J.; Millar, D.; Williams, D. G.

2016-12-01

Sources of water used by plants can differ from those that flow to groundwater and streams. Such ecohydrologic separation forms the basis for the "two water worlds hypothesis" that challenges commonly held notions of how water moves through terrestrial ecosystems. Yet, recent observations in a humid, low energy wetland environment did not support the presence of ecohydrologic separation. These contrasting results, in the context of general physical principles, suggest that energy gradients along the soil-plant-atmosphere continuum may play a role in defining the magnitude of ecohydrologic separation. We quantified ecohydrologic separation in a montane wetland with pronounced hummocks and hollows located in southeastern Wyoming. The rooting zone in this wetland is fully saturated during the spring, but is prone to water table draw-downs (> 1 m) during the summer, likely producing significant water potential differences between plant and soil water pools. We predict that wetland vegetation will express some degree of ecohydrologic separation, but such expression will differ based on microtopgraphic position and the rooting strategy of different plant life form (i.e., trees, shrubs, graminoids). For example, shallowly rooted graminoids on raised hummocks may use water that is distinctly different from that located in wetter hollows, with water in hollows being more isotopically similar to water leaving the wetland via surface water flows. We collected xylem water from dominant plant life forms in hummocks and hollows, free water (via piezometers) and bulk soil water at depths of 20 and 60 cm, as well as surface water and groundwater. Stable isotope ratios of H and O were determined from samples by either laser spectroscopy or isotope ratio mass spectrometry. Our expected results suggest that most of the water being used by wetland plants will be similar to that leaving the wetland via surface flow. In the context of their being two water worlds in the surrounding

Very high pressure liquid chromatography using fully porous particles: quantitative analysis of fast gradient separations without post-run times.

PubMed

Stankovich, Joseph J; Gritti, Fabrice; Stevenson, Paul G; Beaver, Lois Ann; Guiochon, Georges

2014-01-10

Using a column packed with fully porous particles, four methods for controlling the flow rates at which gradient elution runs are conducted in very high pressure liquid chromatography (VHPLC) were tested to determine whether reproducible thermal conditions could be achieved, such that subsequent analyses would proceed at nearly the same initial temperature. In VHPLC high flow rates are achieved, producing fast analyses but requiring high inlet pressures. The combination of high flow rates and high inlet pressures generates local heat, leading to temperature changes in the column. Usually in this case a post-run time is input into the analytical method to allow the return of the column temperature to its initial state. An alternative strategy involves operating the column without a post-run equilibration period and maintaining constant temperature variations for subsequent analysis after conducting one or a few separations to bring the column to a reproducible starting temperature. A liquid chromatography instrument equipped with a pressure controller was used to perform constant pressure and constant flow rate VHPLC separations. Six replicate gradient separations of a nine component mixture consisting of acetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone, and acetanilide dissolved in water/acetonitrile (65:35, v/v) were performed under various experimental conditions: constant flow rate, two sets of constant pressure, and constant pressure operation with a programmed flow rate. The relative standard deviations of the response factors for all the analytes are lower than 5% across the methods. Programming the flow rate to maintain a fairly constant pressure instead of using instrument controlled constant pressure improves the reproducibility of the retention times by a factor of 5, when plotting the chromatograms in time. Copyright © 2013 Elsevier B.V. All rights reserved.

[Determination of unknown impurities in cefotiam hexetil by HPLC-MS/MS].

PubMed

Tang, Qun-Xing; Liu, Ming-Dong; Yan, You-Yi; Ye, Yi; Wang, Zhi-Hui; Zhan, Lan-Fen; Liao, Lin-Chuan

2013-05-01

To detect unknown impurities in raw drug material of cefotiam hexetil. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed for the determination of impurities in cefotiam hexetil. Agilent SB-C18 column (150 mm x 2.1 mm i. d. , 3.5 microm particles) was used for chromatographic separations of cofotiam hexetil dissolved in deionized water, with mobile phase consisting of (A) 0.1% formic acid and (B) acetonitrile and timed gradient program T (min)/B (%): 0/3, 5/3, 15/20, 20/40, 30/60, 40/80. The flow rate was set at 0. 3 mL/min with DAD detector wavelength fixed at 254 nm. Electrospray ionization source was applied and operated in positive ion MRM mode. The source voltage was kept at 4 kV and cone voltage was 100 V with the mass range m/z 50-1000. Nitrogen was used as nebulizing gas and the nebulizer pressure was 40 psi. The drying gas temperature was 350 degrees C and the drying gas flow was 10 L/min. Results Unknown impurities of cefotiam hexetil were identified. Substance 1 was delta3-isomer of cefotiam hexetil. The structures of 3 other substances were also determined. The method is sensitive, rapid and credible for the analysis of cefotiam hexetil and its related impurities, which can be applied in quality control of cefotiam hexetil.

Using contemporary liquid chromatography theory and technology to improve capillary gradient ion-exchange separations.

PubMed

Wouters, Bert; Broeckhoven, Ken; Wouters, Sam; Bruggink, Cees; Agroskin, Yury; Pohl, Christopher A; Eeltink, Sebastiaan

2014-11-28

The gradient-performance limits of capillary ion chromatography have been assessed at maximum system pressure (34.5 MPa) using capillary columns packed with 4.1 μm macroporous anion-exchange particles coated with 65 nm positively-charged nanobeads. In analogy to the van-Deemter curve, the gradient performance was assessed applying different flow rates, while decreasing the gradient time inversely proportional to the increase in flow rate in order to maintain the same retention properties. The gradient kinetic-performance limits were determined at maximum system pressure, applying tG/t0=5, 10, and 20. In addition, the effect of retention on peak width was assessed in gradient mode for mono-, di-, and trivalent inorganic anions. The peak width of late-eluting ions can be significantly reduced by using concave gradient, resulting in better detection sensitivity. A signal enhancement factor of 8 was measured for a late-eluting ion when applying a concave instead of a linear gradient. For the analysis of a complex anion mixture, a coupled column with a total length of 1.05 m was operated at the kinetic-performance limit applying a linear 250 min gradient (tG/t0=10). The peak capacity varied between 200 and 380 depending on analyte retention, and hence on charge and size of the ion. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous determination of nine saponins from Panax notoginseng using HPLC and pressurized liquid extraction.

PubMed

Wan, J B; Lai, C M; Li, S P; Lee, M Y; Kong, L Y; Wang, Y T

2006-04-11

A HPLC and pressurized liquid extraction (PLE) method was developed for simultaneous determination of nine saponins, including notoginsenoside R1, ginsenoside Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3 and Rd in Panax notoginseng. The analysis was performed on C18 column with water-acetonitrile gradient elution and the investigated saponins were authenticated by comparing retention time and mass spectra with their reference compounds. Several methods including PLE, ultrasonication, soxhlet extraction and immersion were used for sample preparation and their extraction efficiency was compared. The results showed that PLE has the highest extraction efficiency and repeatability, which would be valuable on standardization of sample preparation for quality control of Chinese medicines. The developed HPLC and PLE is an effective approach for simultaneously quantitative determination of sapoinins in P. notoginseng, which could be used for quality control of P. notoginseng and its preparations.

Recovery of Manganese Ore Tailings by High-Gradient Magnetic Separation and Hydrometallurgical Method

NASA Astrophysics Data System (ADS)

Zhang, Xiufeng; Tan, Xiumin; Yi, Yuejun; Liu, Weizao; Li, Chun

2017-11-01

With the depletion of high-grade manganese ores, Mn ore tailings are considered valuable secondary resources. In this study, a process combining high-gradient magnetic separation (HGMS) with hydrometallurgical methods is proposed to recycle fine-grained Mn tailings. The Mn tailings were treated by HGMS at 12,500 G to obtain a Mn concentrate of 30% Mn with the recovery efficiency of 64%. The Mn concentrate could be used in the ferromanganese industry. To recover Mn further, the nonmagnetic fraction was leached by SO2 in an H2SO4 solution. Hydrogen peroxide was added to the leachate to oxidize Fe2+ to Fe3+, and the solution pH was adjusted to 5.0-5.5 with ammonia to remove Al, Fe, and Si impurities. The purified solution was reacted with NH4HCO3, and a saleable product of MnCO3 with 97.9% purity was obtained. The combined process can be applied to Mn recovery from finely dispersed weakly magnetic Mn ores or tailings.

An Efficient Method for the Preparative Isolation and Purification of Flavonoids from Leaves of Crataegus pinnatifida by HSCCC and Pre-HPLC.

PubMed

Wen, Lei; Lin, Yunliang; Lv, Ruimin; Yan, Huijiao; Yu, Jinqian; Zhao, Hengqiang; Wang, Xiao; Wang, Daijie

2017-05-09

In this work, flavonoid fraction from the leaves of Crataegus pinnatifida was separated into its seven main constituents using a combination of HSCCC coupled with pre-HPLC. In the first step, the total flavonoid extract was subjected to HSCCC with a two-solvent system of chloroform/methanol/water/ n -butanol (4:3:2:1.5, v/v ), yielding four pure compounds, namely (-)-epicatechin ( 1 ), quercetin-3- O -(2,6-di-α-l-rhamnopyranosyl)-β-d-galactopyranoside ( 2 ), 4''- O -glucosylvitexin ( 3 ) and 2''- O -rhamnosylvitexin ( 4 ) as well as a mixture of three further flavonoids. An extrusion mode was used to rapidly separate quercetin-3- O -(2,6-di-α-l-rhamnopyranosyl)-β-d-galactopyranoside with a big K D -value. In the second step, the mixture that resulted from HSCCC was separated by pre-HPLC, resulting in three pure compounds including: vitexin ( 5 ), hyperoside ( 6 ) and isoquercitrin ( 7 ). The purities of the isolated compounds were established to be over 98%, as determined by HPLC. The structures of these seven flavonoids were elucidated by ESI-MS and NMR spectroscopic analyses.

Superficially porous particles columns for super fast HPLC separations.

PubMed

Ali, Imran; Al-Othman, Zeid A; Al-Za'abi, Mohammed

2012-08-01

Superficially porous silica particles columns (SPSPCs) are manufactured by different companies. The most common have the brand names Halo, Ascentis Express and Kinetex. These columns provide super fast, sharp peaks and moderate sample loading and back pressure. These are available in different chemistries such as C₈, C₁₈, RP Amide and Hilic. Normally, the silica gel particles have 2.7 and 1.7 µm total and inner solid core diameters with 0.5 µm thick outer porous layer, 90 Å pore size and 150 m²/g surface area. They have been used for the separation and identification of low and high molecular weight compounds. The present article describes the state of the art of superficially porous silica particles based columns with special emphasis on their structures, mechanisms of separation, applications and comparison. Copyright © 2012 John Wiley & Sons, Ltd.

Bovine sperm separation by Swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression.

PubMed

Arias, María Elena; Andara, Katherine; Briones, Evelyn; Felmer, Ricardo

2017-06-01

This study assesses the effect of bovine sperm (obtained from three bulls) separation using density gradients (Percoll and BoviPure) and Swim-up on sperm function and gene expression. Sperm evaluations included the plasma membrane integrity (SYBR14/PI), acrosomal integrity (PNA-FITC/PI), oxidative stress (ROS; CH2FDDA), DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (ΔYm; TMRM) using flow cytometry. Sperm motility was evaluated by computer-assisted sperm analysis (CASA) and gene expression using RT-qPCR. The results showed that separation by Percoll achieves a higher proportion of sperm with intact plasma and acrosomal membranes (89.8 and 87.5%, respectively) than the unseparated control (70.3 and 62.4%, respectively), as well as by Swim-up (74.9 and 63.3%, respectively) and BoviPure (83.3 and 80.4%, respectively). No differences were observed in the proportion of spermatozoa with high ΔΨm between Percoll and BoviPure (84.3% and 83.5%, respectively), which were higher than Swim-up and the unseparated control (72.8% and 43.8%, respectively). The ROS levels were higher in the spermatozoa separated by Percoll and no differences were observed in the sperm DNA integrity between all groups. The motility analysis showed that the separation methods improve (p<0.05) total and progressive motility compared to the control, with Percoll proving the most efficient in this regard. Finally, the gene expression analysis of leptin (LEP), aromatase cytochrome P450 (CYP19) and protamine I (PRM1), after validation of 6 reference genes, showed no differences between groups. In conclusion, bovine sperm separation using density gradient improves the parameters of motility and sperm function without affecting the gene expression. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Preparative separation of grape skin polyphenols by high-speed counter-current chromatography.

PubMed

Luo, Lanxin; Cui, Yan; Zhang, Shuting; Li, Lingxi; Li, Yuanyuan; Zhou, Peiyu; Sun, Baoshan

2016-12-01

To develop an efficient method for large preparation of various individual polyphenols from white grape skins (Fernão Pires; Vitis vinifera) by preparative high-speed counter-current chromatography (HSCCC) and preparative-HPLC, an optimized preparative HSCCC condition with two-phase solvent system composed of Hex-EtOAc-H2O (1:50:50, v/v) was used to separate grape skin polyphenols into various fractions. Both the tail-head and head-tail elution modes were used with a flow rate of 3.0ml/min and a rotary speed of 950rpm. Afterwards, a preparative-HPLC separation was applied to isolate individual polyphenols in each of the fractions from HSCCC. Total of 7 fractions (Fraction A to G) were obtained from grape skin extract by HSCCC. After preparative-HPLC isolation, fifteen individual compounds were obtained, most of which presented high yields and purity (all over 90%). The HSCCC method followed with preparative-HPLC appeared to be convenient and economical, constituting an efficient strategy for the isolation of grape skin polyphenols. Copyright © 2016 Elsevier Ltd. All rights reserved.

Determination of 2,4-Dichlorophenoxyacetic acid (2,4-D) in rat serum for pharmacokinetic studies with a simple HPLC method

PubMed Central

Chen, Xiao; Wan, Yanjian; Chen, Xi; Li, Yuanyuan

2018-01-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is a chlorophenoxy herbicide used worldwide. We describe a high-performance liquid chromatography (HPLC) method with UV detection for the determination of 2,4-D in female and male rat serum. This allows to observe the change of serum 2,4-D concentration in rats with time and its pharmacokinetics characteristics with a simple, rapid, optimized and validated method. The serum samples are pretreated and introduced into the HPLC system. The analytes are separated in a XDB-C18 column with a mobile phase of acetonitrile (solvent A) and 0.02 M ammonium acetate (containing 0.1% formic acid) (solvent B) using a gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 230 nm. Calibration curve for 2,4-D was constructed over a range of 0.1–400 mg/L. The method was successfully applied to study the pharmacokinetics of 2,4-D in rats in this study. After oral administration of 300 mg/kg and 60 mg/kg 2,4-D, the mean Cmax values were 601.9 and 218.4 mg/L, the AUC0→∞ values were 23,722 and 4,127 mg×h/L and the clearance (Cl) were 1.10 and 0.02 L/(h×kg), respectively. The developed method was found to be specific, precise, reproducible and rapid. PMID:29342170

Determination of 2,4-Dichlorophenoxyacetic acid (2,4-D) in rat serum for pharmacokinetic studies with a simple HPLC method.

PubMed

Chen, Xiao; Zhang, Hongling; Wan, Yanjian; Chen, Xi; Li, Yuanyuan

2018-01-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is a chlorophenoxy herbicide used worldwide. We describe a high-performance liquid chromatography (HPLC) method with UV detection for the determination of 2,4-D in female and male rat serum. This allows to observe the change of serum 2,4-D concentration in rats with time and its pharmacokinetics characteristics with a simple, rapid, optimized and validated method. The serum samples are pretreated and introduced into the HPLC system. The analytes are separated in a XDB-C18 column with a mobile phase of acetonitrile (solvent A) and 0.02 M ammonium acetate (containing 0.1% formic acid) (solvent B) using a gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 230 nm. Calibration curve for 2,4-D was constructed over a range of 0.1-400 mg/L. The method was successfully applied to study the pharmacokinetics of 2,4-D in rats in this study. After oral administration of 300 mg/kg and 60 mg/kg 2,4-D, the mean Cmax values were 601.9 and 218.4 mg/L, the AUC0→∞ values were 23,722 and 4,127 mg×h/L and the clearance (Cl) were 1.10 and 0.02 L/(h×kg), respectively. The developed method was found to be specific, precise, reproducible and rapid.

[HPLC Fingerprint of QingGuangAn and Determination of the Main Components].

PubMed

Wang, Min; Shen, Bing-bing; Luo, Juan; Chen, Yang; Yang, Yu-pei; Chen, Sheng-huang

2015-10-01

To establish an HPLC fingerprint of ethanol extract of QingGuangAn, and to determine the contents of paeoniflorin and calycosin-7-glucosid. HPLC analysis was performed on an Agilent 1260 Infinity LC system and carried out at 35 degrees C on a column of GRACE Alltima C18 (250 mm x 4.6 mm, 5 μm). A binary gradient elution system was composed of acetonitrile (phase A) and water solution (phase B). Detection was performed at the wavelength of 254 nm, the mobile flow rate was 0.8 mL/min. A matrix including 20 variations (characteristic peaks area) and 10 samples was constructed for similarity evaluation. The results showed that the collected samples had a good similarity. A specificity fingerprint was produced and 20 characteristic peaks were designated. The content of paeoniflorin and calycosin-7-glucosid was 0.368 and 0.049 mg/g, respectively. It is a reliable, available and quick method for quality control of QingGuangAn,which provides some reference for the comparison of different extracting methods of QingGuangAn and the differences of pharmacodynamic.

Industrial application of green chromatography--I. Separation and analysis of niacinamide in skincare creams using pure water as the mobile phase.

PubMed

Yang, Yu; Strickland, Zackary; Kapalavavi, Brahmam; Marple, Ronita; Gamsky, Chris

2011-03-15

In this work, chromatographic separation of niacin and niacinamide using pure water as the sole component in the mobile phase has been investigated. The separation and analysis of niacinamide have been optimized using three columns at different temperatures and various flow rates. Our results clearly demonstrate that separation and analysis of niacinamide from skincare products can be achieved using pure water as the eluent at 60°C on a Waters XTerra MS C18 column, a Waters XBridge C18 column, or at 80°C on a Hamilton PRP-1 column. The separation efficiency, quantification quality, and analysis time of this new method are at least comparable with those of the traditional HPLC methods. Compared with traditional HPLC, the major advantage of this newly developed green chromatography technique is the elimination of organic solvents required in the HPLC mobile phase. In addition, the pure water chromatography separations described in this work can be directly applied in industrial plant settings without further modification of the existing HPLC equipment. Copyright © 2011 Elsevier B.V. All rights reserved.

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog.

PubMed

Dunphy, Michael J; Sysel, Annette M; Lupica, Joseph A; Griffith, Kristie; Sherrod, Taylor; Bauer, Joseph A

2014-04-01

Nitrosylcobalamin (NO-Cbl), a novel vitamin B 12 analog and anti-tumor agent, functions as a biologic 'Trojan horse', utilizing the vitamin B 12 transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. a stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an ascentis ® RP-amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min -1 ) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B 12 therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog

PubMed Central

Dunphy, Michael J.; Sysel, Annette M.; Lupica, Joseph A.; Griffith, Kristie; Sherrod, Taylor

2014-01-01

Nitrosylcobalamin (NO-Cbl), a novel vitamin B12 analog and anti-tumor agent, functions as a biologic ‘Trojan horse’, utilizing the vitamin B12 transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. a stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an ascentis® RP-amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min−1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B12 therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl. PMID:24855323

A Novel HPLC Method for the Concurrent Analysis and Quantitation of Seven Water-Soluble Vitamins in Biological Fluids (Plasma and Urine): A Validation Study and Application

PubMed Central

Grotzkyj Giorgi, Margherita; Howland, Kevin; Martin, Colin; Bonner, Adrian B.

2012-01-01

An HPLC method was developed and validated for the concurrent detection and quantitation of seven water-soluble vitamins (C, B1, B2, B5, B6, B9, B12) in biological matrices (plasma and urine). Separation was achieved at 30°C on a reversed-phase C18-A column using combined isocratic and linear gradient elution with a mobile phase consisting of 0.01% TFA aqueous and 100% methanol. Total run time was 35 minutes. Detection was performed with diode array set at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples (24 plasma and urine samples from abstinent alcohol-dependent males). Interday and intraday precision were <4% and <7%, respectively, for all vitamins. Recovery percentages ranged from 93% to 100%. PMID:22536136

A novel HPLC method for the concurrent analysis and quantitation of seven water-soluble vitamins in biological fluids (plasma and urine): a validation study and application.

PubMed

Giorgi, Margherita Grotzkyj; Howland, Kevin; Martin, Colin; Bonner, Adrian B

2012-01-01

An HPLC method was developed and validated for the concurrent detection and quantitation of seven water-soluble vitamins (C, B(1), B(2), B(5), B(6), B(9), B(12)) in biological matrices (plasma and urine). Separation was achieved at 30°C on a reversed-phase C18-A column using combined isocratic and linear gradient elution with a mobile phase consisting of 0.01% TFA aqueous and 100% methanol. Total run time was 35 minutes. Detection was performed with diode array set at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples (24 plasma and urine samples from abstinent alcohol-dependent males). Interday and intraday precision were <4% and <7%, respectively, for all vitamins. Recovery percentages ranged from 93% to 100%.

Pharmacokinetics of ammonium sulfate gradient loaded liposome-encapsulated oxymorphone and hydromorphone in healthy dogs

PubMed Central

Smith, Lesley J.; Kukanich, Butch K.; Krugner-Higby, Lisa A.; Schmidt, Brynn H.; Heath, Timothy D.

2013-01-01

Objective To evaluate the pharmacokinetics, in dogs, of liposome-encapsulated oxymorphone and hydromorphone made by the ammonium sulfate gradient loading technique (ASG). Animals Four healthy purpose-bred Beagles aged 9.5 ± 3.2 months and weighing 13.4 ± 2.3 kg. Study Design Randomized cross-over design. Methods Each dog was given either 4.0 mg kg−1 of ASG-oxymorphone or 8.0 mg kg−1 of ASG-hydromorphone SC on separate occasions with a 3-month washout period. Blood was collected at baseline and at serial time points up to 1032 hours (43 days) after injection for determination of serum opioid concentrations. Serum opioid concentrations were measured with HPLC-MS and pharmacokinetic parameters were calculated using commercial software and non-compartmental methods. Results Serum concentrations of oxymorphone remained above the limit of quantification for 21 days, while those for hydromorphone remained above the limit of quantification for 29 days. Cmax for ASG-oxymorphone was 7.5 ng mL−1; Cmax for ASG-hydromorphone was 5.7 ng mL−1. Conclusions and clinical relevance Oxymorphone and hydromorphone, when encapsulated into liposomes using the ammonium sulfate gradient loading technique, result in measureable serum concentrations for between 3 to 4 weeks. This formulation may have promise in the convenient use of opioids for clinical treatment of chronically painful conditions in dogs. PMID:23601353

Rapid separation and identification of phenolics in crude red grape skin extracts by high performance liquid chromatography coupled to diode array detection and tandem mass spectrometry.

PubMed

Ji, Mei; Li, Chen; Li, Qiang

2015-10-02

A rapid and efficient method was established for the simultaneous determination of structures and configurations for 45 phenolics isolated from crude red grape skin extracts without extensive sample preparation. Separation and compound assignments were achieved using high performance liquid chromatography coupled to diode array detection and tandem mass spectrometry (HPLC-DAD-MS(2)). A Poroshell 120 EC-C18 (100mm×3.0mm, 2.7μm) column was employed to separate the phenolics, which were eluted using a gradient of acetonitrile and water acidified with 0.2% formic acid. Phenolics were identified by comparison of their UV-vis spectra, mass spectra and MS(2) data with those in the literature. Using this procedure, five compounds were detected for the first time in Vitis amurensis. Good separation of most phenolics was achieved in 26min. The methods described here can be used for the characterization of phenolics in a variety of grapes and grape products. Copyright © 2015 Elsevier B.V. All rights reserved.

Analytical nuclear gradients for the range-separated many-body dispersion model of noncovalent interactions.

PubMed

Blood-Forsythe, Martin A; Markovich, Thomas; DiStasio, Robert A; Car, Roberto; Aspuru-Guzik, Alán

2016-03-01

An accurate treatment of the long-range electron correlation energy, including van der Waals (vdW) or dispersion interactions, is essential for describing the structure, dynamics, and function of a wide variety of systems. Among the most accurate models for including dispersion into density functional theory (DFT) is the range-separated many-body dispersion (MBD) method [A. Ambrosetti et al. , J. Chem. Phys. , 2014, 140 , 18A508], in which the correlation energy is modeled at short-range by a semi-local density functional and at long-range by a model system of coupled quantum harmonic oscillators. In this work, we develop analytical gradients of the MBD energy with respect to nuclear coordinates, including all implicit coordinate dependencies arising from the partitioning of the charge density into Hirshfeld effective volumes. To demonstrate the efficiency and accuracy of these MBD gradients for geometry optimizations of systems with intermolecular and intramolecular interactions, we optimized conformers of the benzene dimer and isolated small peptides with aromatic side-chains. We find excellent agreement with the wavefunction theory reference geometries of these systems (at a fraction of the computational cost) and find that MBD consistently outperforms the popular TS and D3(BJ) dispersion corrections. To demonstrate the performance of the MBD model on a larger system with supramolecular interactions, we optimized the C 60 @C 60 H 28 buckyball catcher host-guest complex. In our analysis, we also find that neglecting the implicit nuclear coordinate dependence arising from the charge density partitioning, as has been done in prior numerical treatments, leads to an unacceptable error in the MBD forces, with relative errors of ∼20% (on average) that can extend well beyond 100%.

[Determination of triterpenoic acids in fruits of Ziziphus jujuba using HPLC-MS with polymeric ODS column].

PubMed

Zhang, Yong; Zhou, An; Xie, Xiao-Mei

2013-03-01

A simple and sensitive method has been developed to simultaneously determine betunilic acid, oleanolic acid and ursolic acid in the fruits of Ziziphus jujuba from different regions by HPLC-MS. This HPLC assay was performed on PAH polymeric C18 bonded stationary phase column with mobile phase contained acetonitrile-water (90: 10) and with negative ESI detection mode. The developed approach was characterized by short time consumption for chromatographic separation, high sensitivity and good reliability so as to meet the requirements for rapid analysis of large-batch fruits of Z. jujuba from different habitats.

[Spectrum-effect Relationship Between Total Antioxidant Activity and HPLC Fingerprint of Arctium lappa Root].

PubMed

Wang, Xiao-juan; Jiang, Lin

2014-12-01

To explore the spectrum-effect relationship between the HPLC fingerprint of Arctium lappa root methanol extract and the total antioxidant activity. The experiment was carried out with Gemini C18 110A (250 mm x 4.6 mm, 5 µm) column using methanol-0.04% phosphoric acid as gradient mobile phase at the flow rate of 1.0 mL/min, detection wavelength of 320 nm. The total antioxidant activity was determined by measuring the absorbance of each sample after being reacted with ammonium molybdate reagent. The spectrum-effect relationship was investigated using canonical correlation analysis (CCA). The spectrum-effect relationship between the HPLC fingerprint of Arctium lappa root methanol extract and the total antioxidant activity were established, the similarity of fingerprint of all samples was above 0.9. Peaks 1, 6, 9, 12 and 14 were principle components of Arctium lappa root for the total antioxidant activity. This method contributes to the fast comprehensive evaluation of quality of Arctium lappa root.

[Simultaneous determination of five cold medicine ingredients in paracetamol triprolidine hydrochloride and pseudoephedrine hydrochloride tablets by pH/organic solvent double-gradient high performance liquid chromatography].

PubMed

Xuan, Xueyi; Huang, Lina; Pan, Xiaoling; Li, Ning

2013-02-01

A pH/organic solvent double-gradient mode in reversed-phase high performance liquid chromatography (HPLC) has been established as a new approach to the simultaneous determination of acetaminophen, caffeine, salicylamide, pseudoephedrine hydrochloride and triprolidine hydrochloride in paracetamol triprolidine hydrochloride and pseudoephedrine hydrochloride tablets. Through the optimization of the organic solvent gradient mode and pH/organic solvent double-gradient mode, the optimum double-gradient HPLC system of the five cold medicine ingredients has been built. The determination was carried out on a Diamonsiol C18 column (250 mm x 4.6 mm, 5 microm). The mobile phase consisted of methanol, 0.05 mol/L ammonium acetate solution and 0.08 mol/L acetic acid solution. The column temperature was set at 30 degrees C. The flow rate was 1.0 mL/min. The sample was measured at multiple wavelengths: 0-6 min, 280 nm; 6-7 min, 257 nm; 7-14 min, 280 nm; 14 min, 233 nm. The separation of the five cold medicine ingredients in the tablets was achieved in 25.5 min. The linear ranges of acetaminophen, pseudoephedrine hydrochloride, caffeine, salicylamide and triprolidine hydrochloride were 0.055 -0.998 g/L, 0.053-0.946 g/L, 0.007-0.129 g/L, 0.035-0.622 g/L and 0.002-0.039 g/L, respectively, with their correlation coefficients greater than 0.999 0. The detection limits (S/N = 3) were 0.09, 6, 0.02, 0.128 and 0.02 mg/L, respectively. Their mean recoveries were 97.9%-102.8%. The advantage of the method is the simultaneous determination of acidic, neutral and basic compounds. It also can improve the column efficiency of the analyte, compress the half-peak width and reduce the trailing. The optimized and validated method can be used for the simultaneous determination of the five cold medicine ingredients in the tablets.

Effect of modulator sorption on gradient shape in ion-exchange chromatography

NASA Technical Reports Server (NTRS)

Velayudhan, A.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

1995-01-01

Mobile phase additives, or modulators, are used in gradient elution chromatography to facilitate separation and reduce separation time. The modulators are usually assumed to be linearly adsorbed or unadsorbed. Here, the consequences of nonlinear modulator adsorption are examined for ion-exchange gradient elution through a series of simulations. Even when the buffer salt is identical to the modulator salt, gradient deformation is observed; the extent of deformation increases as the volume of the feed is increased. When the modulator salt is different from the buffer salt, unusual effects are observed, and the chromatograms are quite different from those predicted by classical gradient elution theory. In particular, local increases in the buffer concentration are found between feed bands, and serve to improve the separation. These effects become more pronounced as the feed volume increases, and could therefore prove valuable in preparative applications.

Crosswind Shear Gradient Affect on Wake Vortices

NASA Technical Reports Server (NTRS)

Proctor, Fred H.; Ahmad, Nashat N.

2011-01-01

Parametric simulations with a Large Eddy Simulation (LES) model are used to explore the influence of crosswind shear on aircraft wake vortices. Previous studies based on field measurements, laboratory experiments, as well as LES, have shown that the vertical gradient of crosswind shear, i.e. the second vertical derivative of the environmental crosswind, can influence wake vortex transport. The presence of nonlinear vertical shear of the crosswind velocity can reduce the descent rate, causing a wake vortex pair to tilt and change in its lateral separation. The LES parametric studies confirm that the vertical gradient of crosswind shear does influence vortex trajectories. The parametric results also show that vortex decay from the effects of shear are complex since the crosswind shear, along with the vertical gradient of crosswind shear, can affect whether the lateral separation between wake vortices is increased or decreased. If the separation is decreased, the vortex linking time is decreased, and a more rapid decay of wake vortex circulation occurs. If the separation is increased, the time to link is increased, and at least one of the vortices of the vortex pair may have a longer life time than in the case without shear. In some cases, the wake vortices may never link.

HPLC studio: a novel software utility to perform HPLC chromatogram comparison for screening purposes.

PubMed

García, J B; Tormo, José R

2003-06-01

A new tool, HPLC Studio, was developed for the comparison of high-performance liquid chromatography (HPLC) chromatograms from microbial extracts. The new utility makes it possible to create a virtual chromatogram by mixing up to 20 individual chromatograms. The virtual chromatogram is the first step in establishing a ranking of the microbial fermentation conditions based on either the area or diversity of HPLC peaks. The utility was used to maximize the diversity of secondary metabolites tested from a microorganism and therefore increase the chances of finding new lead compounds in a drug discovery program.

Comprehensive two-dimensional liquid chromatography separations of pharmaceutical samples using dual Fused-Core columns in the 2nd dimension.

PubMed

Alexander, Anthony J; Ma, Lianjia

2009-02-27

This paper focuses on the application of RPLC x RPLC to pharmaceutical analysis and addresses the specific problem of separating co-eluting impurities/degradation products that maybe "hidden" within the peak envelope of the active pharmaceutical ingredient (API) and thus may escape detection by conventional methods. A comprehensive two-dimensional liquid chromatograph (LC x LC) was constructed from commercially available HPLC equipment. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30s total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates. The operation of the interface is demonstrated for the analysis of a 1mg/ml standard mixture containing 0.05% of a minor component. The practicality of using RPLC x RPLC for the analysis of actual co-eluting pharmaceutical degradation products, by exploiting pH-induced changes in selectivity, is also demonstrated using a three component mixture. This mixture (an API, an oxidation product of the API at 1.0%, w/w, and a photo degradant of the API at 0.5%, w/w) was used to assess the stability indicating nature of an established LC method for analysis of the API.

[HPLC fingerprint of flavonoids in Sophora flavescens and determination of five components].

PubMed

Ma, Hong-Yan; Zhou, Wan-Shan; Chu, Fu-Jiang; Wang, Dong; Liang, Sheng-Wang; Li, Shao

2013-08-01

A simple and reliable method of high-performance liquid chromatography with photodiode array detection (HPLC-DAD) was developed to evaluate the quality of a traditional Chinese medicine Sophora flavescens through establishing chromatographic fingerprint and simultaneous determination of five flavonoids, including trifolirhizin, maackiain, kushenol I, kurarinone and sophoraflavanone G. The optimal conditions of separation and detection were achieved on an ULTIMATE XB-C18 column (4.6 mm x 250 mm, 5 microm) with a gradient of acetonitrile and water, detected at 295 nm. In the chromatographic fingerprint, 13 peaks were selected as the characteristic peaks to assess the similarities of different samples collected from different origins in China according to similarity evaluation for chromatographic fingerprint of traditional chinese medicine (2004AB) and principal component analysis (PCA) were used in data analysis. There were significant differences in the fingerprint chromatograms between S. flavescens and S. tonkinensis. Principal component analysis showed that kurarinone and sophoraflavanone G were the most important component. In quantitative analysis, the five components showed good regression (R > 0.999) with linear ranges, and their recoveries were in the range of 96.3% - 102.3%. This study indicated that the combination of quantitative and chromatographic fingerprint analysis can be readily utilized as a quality control method for S. flavescens and its related traditional Chinese medicinal preparations.

Solvent viscosity mismatch between the solute plug and the mobile phase: Considerations in the applications of two-dimensional HPLC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shalliker, R. Andrew; Guiochon, Georges A

Understanding the nature of viscosity contrast induced flow instabilities is an important aspect in the design of two-dimensional HPLC separations. When the viscosity contrast between the sample plug and the mobile phase is sufficiently large, the phenomenon known as viscous fingering can be induced. Viscous fingering is a flow instability phenomenon that occurs at the interface between two fluids with different viscosities. In liquid chromatography, viscous fingering results in the solute band undergoing a change in form as it enters into the chromatography column. Moreover, even in the absence of viscous fingering, band shapes change shape at low viscosity contrasts.more » These changes can result in a noticeable change in separation performance, with the result depending on whether the solvent pushing the solute plug has a higher or lower viscosity than the solute plug. These viscosity induced changes become more important as the solute injection volume increases and hence understanding the process becomes critical in the implementation of multidimensional HPLC techniques, since in these techniques the sample injection plug into the second dimension is an order of magnitude greater than in one-dimensional HPLC. This review article assesses the current understanding of the viscosity contrast induced processes as they relate to liquid chromatographic separation behaviour.« less

Development and Validation of a Precise, Single HPLC Method for the Determination of Tolperisone Impurities in API and Pharmaceutical Dosage Forms.

PubMed

Raju, Thummala Veera Raghava; Seshadri, Raja Kumar; Arutla, Srinivas; Mohan, Tharlapu Satya Sankarsana Jagan; Rao, Ivaturi Mrutyunjaya; Nittala, Someswara Rao

2013-01-01

A novel, sensitive, stability-indicating HPLC method has been developed for the quantitative estimation of Tolperisone-related impurities in both bulk drugs and pharmaceutical dosage forms. Effective chromatographic separation was achieved on a C18 stationary phase with a simple mobile phase combination delivered in a simple gradient programme, and quantitation was by ultraviolet detection at 254 nm. The mobile phase consisted of a buffer and acetonitrile delivered at a flow rate 1.0 ml/min. The buffer consisted of 0.01 M potassium dihydrogen phosphate with the pH adjusted to 8.0 by using diethylamine. In the developed HPLC method, the resolution between Tolperisone and its four potential impurities was found to be greater than 2.0. Regression analysis showed an R value (correlation coefficient) of greater than 0.999 for the Tolperisone impurities. This method was capable of detecting all four impurities of Tolperisone at a level of 0.19 μg/mL with respect to the test concentration of 1000 μg/mL for a 10 µl injection volume. The tablets were subjected to the stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation. Considerable degradation was found to occur in base hydrolysis, water hydrolysis, and oxidation. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 100%. The established method was validated and found to be linear, accurate, precise, specific, robust, and rugged.

Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde, HPLC and linear discriminant analysis.

PubMed

Beneito-Cambra, M; Bernabé-Zafón, V; Herrero-Martínez, J M; Simó-Alfonso, E F; Ramis-Ramos, G

2009-07-15

The enzymes present in raw materials of the cleaning industry (enzyme industrial concentrates) and in household cleaners were isolated by precipitation with acetone and hydrolyzed with HCl. The resulting amino acids were derivatized with o-phthaldialdehyde, and the derivatives were separated by HPLC. The peaks of 14 amino acids were observed using a C18 column and a multi-segmented gradient of acetonitrile-water in the presence of a 5 mM citric/citrate buffer of pH 6.5. Using either normalized peak areas (divided by the sum of the peak areas of the chromatogram) or ratios of pairs of peak areas as predictor variables, linear discriminant analysis models, capable of predicting the enzyme class, including proteases, lipases, amylases and cellulases, were constructed. For this purpose, both enzyme industrial concentrates and detergent bases spiked with them were included in the training set. In all cases, the enzymes of the evaluation set, including industrial concentrates, spiked detergent bases and commercial cleaners were correctly classified with assignment probabilities higher than 99%.

[HPLC fingerprint chromatogram of Polygonum multiflorum from Guizhou].

PubMed

Li, Yan; Wang, Hui-Juan; Lin, Bing; Zhao, Zhi; Zhou, Ying

2012-12-01

To establish the fingerprint of Polygonum multiflorum from Guizhou and provide a standard for its quality control. HPLC analysis was performed on Agillent ZABAX-C18 (4.6 mm x 250 mm, 5 microm), gradient eluted composed of acetonitrile-0.4% water solution of phosphoric acid. Column temperature was set at 25 degrees C and the flow rate was 1 mL/min. The detection wavelength was 280 nm and the analysis time was 60 min. 9 common peaks were identified. The RSD of the relative retention time and the relative peak area were less than 3% in analyzing its precision, stability and repeatability of the common peaks, and the similarity of the 16 batches of sample was more than 0.9. The method is simple and reliable, and it can provide a standard and guidance for quality control of Polygonum multiflorum.

Separation and purification of bioactive botrallin and TMC-264 by a combination of HSCCC and semi-preparative HPLC from endophytic fungus Hyalodendriella sp. Ponipodef12.

PubMed

Mao, Ziling; Luo, Ruiya; Luo, Haiyu; Tian, Jin; Liu, Hongwei; Yue, Yang; Wang, Mingan; Peng, Youliang; Zhou, Ligang

2014-09-01

Two dibenzo-α-pyrones, botrallin (1) and TMC-264 (2) were preparatively separated from crude ethyl acetate extract of the endophytic fungus Hyalodendriella sp. Ponipodef12, which was isolated from the hybrid 'Neva' of Populus deltoides Marsh × P. nigra L. using a combination of high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC. Botrallin (1) with 74.73% of purity and TMC-264 (2) with 82.29% of purity were obtained through HSCCC by employing a solvent system containing n-hexane-ethyl acetate-methanol-water at a volume ratio of 1.2:1.0:0.9:1.0. It was the first time for TMC-264 (2) to be isolated from this fungus. TMC-264 (2) showed strong antimicrobial and antinematodal activity, and botrallin (1) exhibited moderate inhibitory activity on acetylcholinesterase.

Enhancing Centrifugal Separation With Electrophoresis

NASA Technical Reports Server (NTRS)

Herrmann, F. T.

1986-01-01

Separation of biological cells by coil-planet centrifuge enhanced by electrophoresis. By itself, coil-planet centrifuge offers relatively gentle method of separating cells under low centrifugal force in physiological medium that keeps cells alive. With addition of voltage gradient to separation column of centrifuge, separation still gentle but faster and more complete. Since separation apparatus contains no rotary seal, probability of leakage, contamination, corrosion, and short circuits reduced.

Multidimensional Separation of Natural Products Using Liquid Chromatography Coupled to Hadamard Transform Ion Mobility Mass Spectrometry

NASA Astrophysics Data System (ADS)

Liu, Wenjie; Zhang, Xing; Knochenmuss, Richard; Siems, William F.; Hill, Herbert H.

2016-05-01

A high performance liquid chromatograph (HPLC)was interfaced to an atmospheric drift tube ion mobility time of flight mass spectrometry. The power of multidimensional separation was demonstrated using chili pepper extracts. The ambient pressure drift tube ion mobility provided high resolving powers up to 166 for the HPLC eluent. With implementation of Hadamard transform (HT), the duty cycle for the ion mobility drift tube was increased from less than 1% to 50%, and the ion transmission efficiency was improved by over 200 times compared with pulsed mode, improving signal to noise ratio 10 times. HT ion mobility and TOF mass spectrometry provide an additional dimension of separation for complex samples without increasing the analysis time compared with conventional HPLC.

Simulation of elution profiles in liquid chromatography - II: Investigation of injection volume overload under gradient elution conditions applied to second dimension separations in two-dimensional liquid chromatography.

PubMed

Stoll, Dwight R; Sajulga, Ray W; Voigt, Bryan N; Larson, Eli J; Jeong, Lena N; Rutan, Sarah C

2017-11-10

An important research direction in the continued development of two-dimensional liquid chromatography (2D-LC) is to improve the detection sensitivity of the method. This is especially important in applications where injection of large volumes of effluent from the first dimension ( 1 D) column into the second dimension ( 2 D) column leads to severe 2 D peak broadening and peak shape distortion. For example, this is common when coupling two reversed-phase columns and the organic solvent content of the 1 D mobile phase overwhelms the 2 D column with each injection of 1 D effluent, leading to low resolution in the second dimension. In a previous study we validated a simulation approach based on the Craig distribution model and adapted from the work of Czok and Guiochon [1] that enabled accurate simulation of simple isocratic and gradient separations with very small injection volumes, and isocratic separations with mismatched injection and mobile phase solvents [2]. In the present study we have extended this simulation approach to simulate separations relevant to 2D-LC. Specifically, we have focused on simulating 2 D separations where gradient elution conditions are used, there is mismatch between the sample solvent and the starting point in the gradient elution program, injection volumes approach or even exceed the dead volume of the 2 D column, and the extent of sample loop filling is varied. To validate this simulation we have compared results from simulations and experiments for 101 different conditions, including variation in injection volume (0.4-80μL), loop filling level (25-100%), and degree of mismatch between sample organic solvent and the starting point in the gradient elution program (-20 to +20% ACN). We find that that the simulation is accurate enough (median errors in retention time and peak width of -1.0 and -4.9%, without corrections for extra-column dispersion) to be useful in guiding optimization of 2D-LC separations. However, this requires that real

HPLC separation of acetaminophen and its impurities using a mixed-mode reversed-phase/cation exchange stationary phase.

PubMed

Călinescu, Octavian; Badea, Irinel A; Vlădescu, Luminiţa; Meltzer, Viorica; Pincu, Elena

2012-04-01

Determination of acetaminophen and its main impurities: 4-nitrophenol, 4'-chloroacetanilide, as well as 4-aminophenol and its degradation products, p-benzoquinone and hydroquinone has been developed and validated by a new high-performance liquid chromatography method. Chromatographic separation has been obtained on a Hypersil Duet C18/SCX column, using gradient elution, with a mixture of phosphate buffer (pH = 4.88) and methanol as a mobile phase. Analysis time did not exceed 14.5 min and good resolutions, peak shapes and asymmetries have resulted. The linearity of the method has been tested in the range of 5.0-60 µg/mL for acetaminophen and 0.5-6 µg/mL for the other compounds. The limits of detection and quantification have been also established to be lower than 0.1 µg/mL and 0.5 µg/mL, respectively. The method has been successfully applied for the analysis of commercial acetaminophen preparations. © The Author [2012]. Published by Oxford University Press. All rights reserved.

Multiphase Transport in Porous Media: Gas-Liquid Separation Using Capillary Pressure Gradients International Space Station (ISS) Flight Experiment Development

NASA Technical Reports Server (NTRS)

Wheeler, Richard R., Jr.; Holtsnider, John T.; Dahl, Roger W.; Deeks, Dalton; Javanovic, Goran N.; Parker, James M.; Ehlert, Jim

2013-01-01

Advances in the understanding of multiphase flow characteristics under variable gravity conditions will ultimately lead to improved and as of yet unknown process designs for advanced space missions. Such novel processes will be of paramount importance to the success of future manned space exploration as we venture into our solar system and beyond. In addition, because of the ubiquitous nature and vital importance of biological and environmental processes involving airwater mixtures, knowledge gained about fundamental interactions and the governing properties of these mixtures will clearly benefit the quality of life here on our home planet. The techniques addressed in the current research involving multiphase transport in porous media and gas-liquid phase separation using capillary pressure gradients are also a logical candidate for a future International Space Station (ISS) flight experiment. Importantly, the novel and potentially very accurate Lattice-Boltzmann (LB) modeling of multiphase transport in porous media developed in this work offers significantly improved predictions of real world fluid physics phenomena, thereby promoting advanced process designs for both space and terrestrial applications.This 3-year research effort has culminated in the design and testing of a zero-g demonstration prototype. Both the hydrophilic (glass) and hydrophobic (Teflon) media Capillary Pressure Gradient (CPG) cartridges prepared during the second years work were evaluated. Results obtained from ground testing at 1-g were compared to those obtained at reduced gravities spanning Martian (13-g), Lunar (16-g) and zero-g. These comparisons clearly demonstrate the relative strength of the CPG phenomena and the efficacy of its application to meet NASAs unique gas-liquid separation (GLS) requirements in non-terrestrial environments.LB modeling software, developed concurrently with the zero-g test effort, was shown to accurately reproduce observed CPG driven gas-liquid separation

Application of the artificial neural network in quantitative structure-gradient elution retention relationship of phenylthiocarbamyl amino acids derivatives.

PubMed

Tham, S Y; Agatonovic-Kustrin, S

2002-05-15

Quantitative structure-retention relationship(QSRR) method was used to model reversed-phase high-performance liquid chromatography (RP-HPLC) separation of 18 selected amino acids. Retention data for phenylthiocarbamyl (PTC) amino acids derivatives were obtained using gradient elution on ODS column with mobile phase of varying acetonitrile, acetate buffer and containing 0.5 ml/l of triethylamine (TEA). Molecular structure of each amino acid was encoded with 36 calculated molecular descriptors. The correlation between the molecular descriptors and the retention time of the compounds in the calibration set was established using the genetic neural network method. A genetic algorithm (GA) was used to select important molecular descriptors and supervised artificial neural network (ANN) was used to correlate mobile phase composition and selected descriptors with the experimentally derived retention times. Retention time values were used as the network's output and calculated molecular descriptors and mobile phase composition as the inputs. The best model with five input descriptors was chosen, and the significance of the selected descriptors for amino acid separation was examined. Results confirmed the dominant role of the organic modifier in such chromatographic systems in addition to lipophilicity (log P) and molecular size and shape (topological indices) of investigated solutes.

Hand-Portable Gradient Capillary Liquid Chromatography Pumping System.

PubMed

Sharma, Sonika; Plistil, Alex; Barnett, Hal E; Tolley, H Dennis; Farnsworth, Paul B; Stearns, Stanley D; Lee, Milton L

2015-10-20

In this work, a novel splitless nanoflow gradient generator integrated with a stop-flow injector was developed and evaluated using an on-column UV-absorption detector. The gradient pumping system consisted of two nanoflow pumps controlled by micro stepper motors, a mixer connected to a serpentine tube, and a high-pressure valve. The gradient system weighed only 4 kg (9 lbs) and could generate up to 55 MPa (8000 psi) pressure. The system could operate using a 24 V DC battery and required 1.2 A for operation. The total volume capacity of the pump was 74 μL, and a sample volume of 60 nL could be injected. The system provided accurate nanoflow rates as low as 10 nL/min without employing a splitter, making it ideal for capillary column use. The gradient dwell volume was calculated to be 1.3 μL, which created a delay of approximately 4 min with a typical flow rate of 350 nL/min. Gradient performance was evaluated for gradient step accuracy, and excellent reproducibility was obtained in day-to-day experiments (RSD < 1.2%, n = 4). Linear gradient reproducibility was tested by separating a three-component pesticide mixture on a poly(ethylene glycol) diacrylate (PEGDA) monolithic column. The retention time reproducibility was very good in run-to-run experiments (RSD < 1.42%, n = 4). Finally, excellent separation of five phenols was demonstrated using the nanoflow gradient system.

Simultaneous determination of potassium guaiacolsulfonate, guaifenesin, diphenhydramine HCl and carbetapentane citrate in syrups by using HPLC-DAD coupled with partial least squares multivariate calibration.

PubMed

Dönmez, Ozlem Aksu; Aşçi, Bürge; Bozdoğan, Abdürrezzak; Sungur, Sidika

2011-02-15

A simple and rapid analytical procedure was proposed for the determination of chromatographic peaks by means of partial least squares multivariate calibration (PLS) of high-performance liquid chromatography with diode array detection (HPLC-DAD). The method is exemplified with analysis of quaternary mixtures of potassium guaiacolsulfonate (PG), guaifenesin (GU), diphenhydramine HCI (DP) and carbetapentane citrate (CP) in syrup preparations. In this method, the area does not need to be directly measured and predictions are more accurate. Though the chromatographic and spectral peaks of the analytes were heavily overlapped and interferents coeluted with the compounds studied, good recoveries of analytes could be obtained with HPLC-DAD coupled with PLS calibration. This method was tested by analyzing the synthetic mixture of PG, GU, DP and CP. As a comparison method, a classsical HPLC method was used. The proposed methods were applied to syrups samples containing four drugs and the obtained results were statistically compared with each other. Finally, the main advantage of HPLC-PLS method over the classical HPLC method tried to emphasized as the using of simple mobile phase, shorter analysis time and no use of internal standard and gradient elution. Copyright © 2010 Elsevier B.V. All rights reserved.

Development and Optimisation of an HPLC-DAD-ESI-Q-ToF Method for the Determination of Phenolic Acids and Derivatives

PubMed Central

Restivo, Annalaura; Degano, Ilaria; Ribechini, Erika; Colombini, Maria Perla

2014-01-01

A method for the HPLC-MS/MS analysis of phenols, including phenolic acids and naphtoquinones, using an amide-embedded phase column was developed and compared to the literature methods based on classical C18 stationary phase columns. RP-Amide is a recently developed polar embedded stationary phase, whose wetting properties mean that up to 100% water can be used as an eluent. The increased retention and selectivity for polar compounds and the possibility of working in 100% water conditions make this column particularly interesting for the HPLC analysis of phenolic acids and derivatives. In this study, the chromatographic separation was optimised on an HPLC-DAD, and was used to separate 13 standard phenolic acids and derivatives. The method was validated on an HPLC-ESI-Q-ToF. The acquisition was performed in negative polarity and MS/MS target mode. Ionisation conditions and acquisition parameters for the Q-ToF detector were investigated by working on collision energies and fragmentor potentials. The performance of the method was fully evaluated on standards. Moreover, several raw materials containing phenols were analysed: walnut, gall, wine, malbec grape, French oak, red henna and propolis. Our method allowed us to characterize the phenolic composition in a wide range of matrices and to highlight possible matrix effects. PMID:24551158

Development and optimisation of an HPLC-DAD-ESI-Q-ToF method for the determination of phenolic acids and derivatives.

PubMed

Restivo, Annalaura; Degano, Ilaria; Ribechini, Erika; Colombini, Maria Perla

2014-01-01

A method for the HPLC-MS/MS analysis of phenols, including phenolic acids and naphtoquinones, using an amide-embedded phase column was developed and compared to the literature methods based on classical C18 stationary phase columns. RP-Amide is a recently developed polar embedded stationary phase, whose wetting properties mean that up to 100% water can be used as an eluent. The increased retention and selectivity for polar compounds and the possibility of working in 100% water conditions make this column particularly interesting for the HPLC analysis of phenolic acids and derivatives. In this study, the chromatographic separation was optimised on an HPLC-DAD, and was used to separate 13 standard phenolic acids and derivatives. The method was validated on an HPLC-ESI-Q-ToF. The acquisition was performed in negative polarity and MS/MS target mode. Ionisation conditions and acquisition parameters for the Q-ToF detector were investigated by working on collision energies and fragmentor potentials. The performance of the method was fully evaluated on standards. Moreover, several raw materials containing phenols were analysed: walnut, gall, wine, malbec grape, French oak, red henna and propolis. Our method allowed us to characterize the phenolic composition in a wide range of matrices and to highlight possible matrix effects.

Separation of carbon nanotubes into chirally enriched fractions

DOEpatents

Doorn, Stephen K [Los Alamos, NM; Niyogi, Sandip [Los Alamos, NM

2012-04-10

A mixture of single-walled carbon nanotubes ("SWNTs") is separated into fractions of enriched chirality by preparing an aqueous suspension of a mixture of SWNTs and a surfactant, injecting a portion of the suspension on a column of separation medium having a density gradient, and centrifuging the column. In some embodiments, salt is added prior to centrifugation. In other embodiments, the centrifugation is performed at a temperature below room temperature. Fractions separate as colored bands in the column. The diameter of the separated SWNTs decreases with increasing density along the gradient of the column. The colored bands can be withdrawn separately from the column.

Magnetoelectric Transverse Gradient Sensor with High Detection Sensitivity and Low Gradient Noise

PubMed Central

2017-01-01

We report, theoretically and experimentally, the realization of a high detection performance in a novel magnetoelectric (ME) transverse gradient sensor based on the large ME effect and the magnetic field gradient (MFG) technique in a pair of magnetically-biased, electrically-shielded, and mechanically-enclosed ME composites having a transverse orientation and an axial separation. The output voltage of the gradient sensor is directly obtained from the transverse MFG-induced difference in ME voltage between the two ME composites and is calibrated against transverse MFGs to give a high detection sensitivity of 0.4–30.6 V/(T/m), a strong common-mode magnetic field noise rejection rate of <−14.5 dB, a small input-output nonlinearity of <10 ppm, and a low gradient noise of 0.16–620 nT/m/Hz in a broad frequency range of 1 Hz–170 kHz under a small baseline of 35 mm. An analysis of experimental gradient noise spectra obtained in a magnetically-unshielded laboratory environment reveals the domination of the pink (1/f) noise, dielectric loss noise, and power-frequency noise below 3 kHz, in addition to the circuit noise above 3 kHz, in the gradient sensor. The high detection performance, together with the added merit of passive and direct ME conversion by the large ME effect in the ME composites, makes the gradient sensor suitable for the passive, direct, and broadband detection of transverse MFGs. PMID:29068428

Magnetoelectric Transverse Gradient Sensor with High Detection Sensitivity and Low Gradient Noise.

PubMed

Zhang, Mingji; Or, Siu Wing

2017-10-25

We report, theoretically and experimentally, the realization of a high detection performance in a novel magnetoelectric (ME) transverse gradient sensor based on the large ME effect and the magnetic field gradient (MFG) technique in a pair of magnetically-biased, electrically-shielded, and mechanically-enclosed ME composites having a transverse orientation and an axial separation. The output voltage of the gradient sensor is directly obtained from the transverse MFG-induced difference in ME voltage between the two ME composites and is calibrated against transverse MFGs to give a high detection sensitivity of 0.4-30.6 V/(T/m), a strong common-mode magnetic field noise rejection rate of <-14.5 dB, a small input-output nonlinearity of <10 ppm, and a low gradient noise of 0.16-620 nT/m/ Hz in a broad frequency range of 1 Hz-170 kHz under a small baseline of 35 mm. An analysis of experimental gradient noise spectra obtained in a magnetically-unshielded laboratory environment reveals the domination of the pink (1/ f ) noise, dielectric loss noise, and power-frequency noise below 3 kHz, in addition to the circuit noise above 3 kHz, in the gradient sensor. The high detection performance, together with the added merit of passive and direct ME conversion by the large ME effect in the ME composites, makes the gradient sensor suitable for the passive, direct, and broadband detection of transverse MFGs.

A validated HPLC-MS/MS assay for quantifying unstable pharmacologically active metabolites of clopidogrel in human plasma: application to a clinical pharmacokinetic study.

PubMed

Furlong, Michael T; Savant, Ishani; Yuan, Moucun; Scott, Laura; Mylott, William; Mariannino, Thomas; Kadiyala, Pathanjali; Roongta, Vikram; Arnold, Mark E

2013-05-01

Clopidogrel is prescribed for the treatment of Acute Coronary Syndrome and recent myocardial infarction, recent stroke, or established peripheral arterial disease. A sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated to enable reliable quantification of four diastereomeric and chemically reactive thiol metabolites, two of which are pharmacologically active, in human plasma. The metabolites were stabilized by alkylation of their reactive thiol moieties with 2-bromo-3'-methoxyacetophenone (MPB). Following organic solvent mediated-protein precipitation in a 96-well plate format, chromatographic separation was achieved by gradient elution on an Ascentis Express RP-amide column. Chromatographic conditions were optimized to ensure separation of the four derivatized active metabolites. Derivatized metabolites and stable isotope-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The HPLC-MS/MS assay was validated over concentration ranges of 0.125-125 ng/mL for metabolites H1-H3 and 0.101-101 ng/mL for H4. Intra- and inter-assay precision values for replicate quality control samples were within 14.3% for all analytes during the assay validation. Mean quality control accuracy values were within ±6.3% of nominal values for all analytes. Assay recoveries were high (>79%). The four derivatized analytes were stable in human blood for at least 2 h at room temperature and on ice. The analytes were also stable in human plasma for at least 25 h at room temperature, 372 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of all four thiol metabolites in human plasma in support of a human pharmacokinetic study. Copyright © 2013 Elsevier B.V. All rights reserved.

Combination of HPLC chromatogram and hypoglycemic effect identifies isoflavones as the principal active fraction of Belamcanda chinensis leaf extract in diabetes treatment.

PubMed

Chen, Yan; Wu, Chong-Ming; Dai, Rong-Ji; Li, Liang; Yu, Yu-Hong; Li, Yan; Meng, Wei-Wei; Zhang, Liang; Zhang, Yongqian; Deng, Yu-Lin

2011-02-15

In previous study, we demonstrated the hypoglycemic effect of aqueous extract of Belamcanda chinensis leaves in rats. Here, we separated the aqueous extract of B. chinensis leaves and investigated the spectrum-effect relationships between HPLC chromatograms and hypoglycemic activities of different isolates from B. chinensis leaf extract. Sequential solvent extraction with petroleum ether, chloroform, acetic ester and n-butanol provided several isolates showing similar hypoglycemic activities, making it difficult to discriminate the active fractions. Stepwise elution through HP20 macroporous resin by water, 40% and 95% ethanol provided isolates with distinct hypoglycemic activities, representing a simple, rapid and efficient preparative separation method. Combination of HPLC chromatogram and pharmacological effect targeted a hypoglycemic activity-related region in HPLC chromatogram. Each peak in this region was analyzed by UV spectrum scan. Most of them were flavonoids in which tectoridin and swertisin were known flavonoids with anti-diabetic activities. In together, this work provides a general model of combination of HPLC chromatography and pharmacological effect to study the spectrum-effect relationships of aqueous extract from B. chinensis leaves, which can be used to find principle components of B. chinensis on pharmacological activity. Copyright © 2011 Elsevier B.V. All rights reserved.

[Simultaneous separation and detection of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate by RP-HPLC and structure confirmation].

PubMed

Zhao, Yan-Yan; Liu, Li-Yan; Han, Yuan-Yuan; Li, Yue-Qiu; Wang, Yan; Shi, Min-Jian

2013-08-01

A simple, fast and sensitive analytical method for the simultaneous separation and detection of 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B by RP-HPLC and drug quality standard was established. The structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate have been confirmed. Reference European Pharmacopoeia EP7.0 version, British Pharmacopoeia 2012 version, National Drug Standards of China (WS 1-XG-2002), domestic and international interrelated literature were referred to select the composition of mobile phase. The experimental parameters including salt concentration, pH, addition quantities of organic solvent, column temperature and flow rate were optimized. Finally, the assay was conducted on a Durashell-C18 column (250 mm x 4.6 mm, 5 microm) with 0.01 mol x mL(-1) ammonium perchlorate (add ammonia to adjust the pH value to 8.2) -methanol (48 : 52) as mobile phase at the flow rate of 0.8 mL x min(-1), and the detection wavelength was set at 254 nm. The column temperature was 50 degrees C and the injection volume was 10 microL. The MS, NMR, UV and RP-HPLC were used to confirm the structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate. Under the optimized separation conditions, the calibration curves of 18 alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B showed good linearity within the concentration of 0.50-100 microg x mL(-1) (r = 0.999 9). The detection limits for 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B were 0.15, 0.10, 0.10, 0.15 microg x mL(-1) respectively. The method is sensitive, reproducible and the results are accurate and reliable. It can be used for chiral resolution of 18alpha-glycyrrhizinic acid, 18Pbeta-glycyrrhizinic acid, and detection content of principal component and

[Simultaneous determination of eight kinds of conjunct bile acids in human bile by R-HPLC].

PubMed

Dai, Z; Tan, G; Qian, K; Chen, X

1997-01-01

A method for the simultaneous determination of eight kinds of conjunct bile acids in human bile was developed by HPLC. They were separated on a YWG-C18 (3 microns) column at 30 degrees C, with methanol/water (65/35, V/V, pH3.0) as mobile phase, and detection wavelength at UV 210 nm. The linear ranges were 50-1,000 microns.ml-1, the recoveries were 91.2%-108.6%. The biles of 30 cases with cholelithiasis cholecystolithiasis and 20 cases without gallstone were detected by HPLC. The results showed that the constitution of bile acids was different between patients with cholelithiasis cholecystolithiasis and patients without gallstone.

Validation a solid-phase extraction-HPLC method for determining the migration behaviour of five aromatic amines from packaging bags into seafood simulants.

PubMed

OuYang, Xiao-Kun; Luo, Yu-Yang; Wang, Yang-Guang; Yang, Li-Ye

2014-01-01

The simultaneous determination of five aromatic amines and their potential migration from packaging bags into seafood simulants were investigated. A validated HPLC method was developed for the separation and qualification of five aromatic amines in seafood simulants. By combining solid-phase extraction (SPE), these amines were efficiently separated on a Halo C18 column (150 × 4.6 mm i.d., 2.7 μm, particle size) using a mobile phase of methanol/phosphate buffer solution (5 mmol l(-1), pH 6.9) with gradient elution. The linear range was 0.1-10.0 mg l(-1); the absolute recoveries ranged from 85.3% to 98.4%; and the limits of detection of the five aromatic amines were between 0.015 and 0.08 mg l(-1). In this work the migration profile of aromatic amines from black plastic bags was investigated at temperatures of 4°C with water, 3% acetic acid solution, 10% ethanol solution and 50% ethanol solution as seafood simulants, respectively. The migration of the five aromatic amines under different conditions showed that residual o-methoxyaniline, p-chloroaniline, aniline and 2,6-dimethylaniline leaching from black plastic bags increased with incubation time. No detectable 3,3´-dimethylbenzidine was found to leach from the bags.

An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels.

PubMed

Gonçalves, A M; Nehme, N S; Morel, C M

1990-01-01

A simple protocol is described for the silver staining of polyacrylamide gradient gels used for the separation of restriction fragments of kinetoplast DNA [schizodeme analysis of trypanosomatids (Morel et al., 1980)]. The method overcomes the problems of non-uniform staining and strong background color which are frequently encountered when conventional protocols for silver staining of linear gels are applied to gradient gels. The method described has proven to be of general applicability for DNA, RNA and protein separations in gradient gels.

RP-HPLC Determination of Phenylalkanoids and Monoterpenoids in Rhodiola rosea and Identification by LC-ESI-TOF

USDA-ARS?s Scientific Manuscript database

An HPLC method permitting the simultaneous determination of fourteen compounds (phenylalkanoids and monoterpenoids) from the roots of Rhodiola rosea was developed. A separation was achieved within 35 minutes by using C-18 column material, a water/acetonitrile mobile phase, both containing 0.05% phos...

HPLC-MS/MS method for the simultaneous determination of MB07133 and its metabolites, cytarabine and arabinofuranosyluracil, in rat plasma.

PubMed

Wang, Dan; Xiao, Qingqing; Yang, Wanqiu; Qian, Wei; Yang, Jin

2016-02-20

MB07133 is an intravenously administered cytarabine mononucleotide (araCMP) prodrug, for the treatment of hepatocellular carcinoma (HCC). A simple, selective and sensitive HPLC-MS/MS method using high pressure liquid chromatography (HPLC) coupled to triple-quadrupole mass spectrometer, was developed and validated for the detection of prodrug MB07133 and its metabolites, cytarabine (araC) and arabinofuranosyluracil (araU) in rat plasma. Protein precipitation using 3% trichloroacetic acid (TCA) was employed to extract analytes from 100μL rat plasma. Adequate separation of araC and araU from their endogenous compounds was achieved on the Synergi(®) fusion-RP column (150mm×4.6mm, 4μm) by a gradient-elution with a mobile phase consisting of ammonium formate (1mM) and methanol at a flow rate of 1mL/min. Multiple reaction monitoring mode (MRM) was applied in the detection of MB07133, araC, araU and Ganciclovir (internal standard) with ion pairs 441.2/330.2, 244.2/112.2, 245.2/113.2 and 256.1/152.2, respectively. The assays were validated with respect to specificity, linearity (100-50000ng/mL for MB07133, 2-1000ng/mL for araC and araU), accuracy and precision, extraction recovery, matrix effect and stability. The validated method has been successfully applied to an intravenous bolus pharmacokinetic study of MB07133 in male Sprague-Dawley rats (18mg/kg i.v.). Copyright © 2015 Elsevier B.V. All rights reserved.

LIQUID CHROMATOGRAPHIC SEPARATION OF THE ENANTIOMERS OF TRANS-CHLORDANE, CIS-CHLORDANE, HEPTACHLOR, HEPTACHLOR EPOXIDE AND ALPHA-HEXACHLOROCYCLOHEXANE WITH APPLICATION TO SMALL-SCALE PREPARATIVE SEPARATION

EPA Science Inventory

Analytical high-performance liquid chromatographic separations of the individual enantiomers of five polychlorinated compounds were obtained on polysaccharide stereoselective HPLC columns. The enantiomers of the pesticides trans-chlordane, cis-chlordane and heptachlor were separa...

Simultaneous analysis of insect repellent DEET, sunscreen oxybenzone and five relevant metabolites by reversed-phase HPLC with UV detection: application to an in vivo study in a piglet model.

PubMed

Kasichayanula, Sreeneeranj; House, James D; Wang, Tao; Gu, Xiaochen

2005-08-05

N,N-Diethyl-m-toluamide (DEET) and oxybenzone are two essential active ingredients in insect repellent and sunscreen preparations. We developed and validated a simple, sensitive, and selective HPLC assay to simultaneously measure DEET, oxybenzone and five primary metabolites of DEET and oxybenzone in biological samples including plasma, urine and skin strips. The compounds were separated on a reversed-phase C18 column using three-stage gradient steps with methanol and water. DEET and two relevant metabolites were detected at 254 nm, while oxybenzone and three relevant metabolites were detected at 289 nm. The limit of detection was 0.6 ng for DEET and 0.5 ng for oxybenzone, respectively. The developed method was further applied to analyze various biological samples from an in vivo animal study that evaluated concurrent use of commercially available insect repellent and sunscreen preparations.

Comparison of UHPLC and HPLC in Benzodiazepines Analysis of Postmortem Samples

PubMed Central

Behnoush, Behnam; Sheikhazadi, Ardeshir; Bazmi, Elham; Fattahi, Akbar; Sheikhazadi, Elham; Saberi Anary, Seyed Hossein

2015-01-01

Abstract The aim of this study was to compare system efficiency and analysis duration regarding the solvent consumption and system maintenance in high-pressure liquid chromatography (HPLC) and ultra high-pressure liquid chromatography (UHPLC). In a case–control study, standard solutions of 7 benzodiazepines (BZs) and 73 biological samples such as urine, tissue, stomach content, and bile that screened positive for BZs were analyzed by HPLC and UHPLC in laboratory of forensic toxicology during 2012 to 2013. HPLC analysis was performed using a Knauer by 100-5 C-18 column (250 mm × 4.6 mm) and Knauer photodiode array detector (PAD). UHPLC analysis was performed using Knauer PAD detector with cooling autosampler and Eurospher II 100-3 C-18 column (100 mm × 3 mm) and also 2 pumps. The mean retention time, standard deviation, flow rate, and repeatability of analytical results were compared by using 2 methods. Routine runtimes in HPLC and UHPLC took 40 and 15 minutes, respectively. Changes in mobile phase composition of the 2 methods were not required. Flow rate and solvent consumption in UHPLC decreased. Diazepam and flurazepam were detected more frequently in biological samples. In UHPLC, small particle size and short length of column cause effective separation of BZs in a very short time. Reduced flow rate, solvent consumption, and injection volume cause more efficiency and less analysis costs. Thus, in the detection of BZs, UHPLC is an accurate, sensitive, and fast method with less cost of analysis. PMID:25860209

An HPLC method for the determination of selected amino acids in human embryo culture medium.

PubMed

Drábková, Petra; Andrlová, Lenka; Kanďár, Roman

2017-02-01

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP 18e or Ascentis Express C 18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos. Copyright © 2016 John Wiley & Sons, Ltd.

Ellagitannin composition of blackberry as determined by HPLC-ESI-MS and MALDI-TOF-MS.

PubMed

Hager, Tiffany J; Howard, Luke R; Liyanage, Rohana; Lay, Jackson O; Prior, Ronald L

2008-02-13

Blackberries ( Rubus sp.) were evaluated by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) to identify the ellagitannins present in flesh, torus (receptacle tissue), and seeds. Most ellagitannins were present (or detectable) only in seed tissues. Ellagitannins identified by HPLC-ESI-MS in the seeds included pedunculagin, casuarictin/potentillin, castalagin/vescalagin, lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D. For several of the ellagitannins, isomeric separation was also obtained. The MALDI-TOF-MS analysis was primarily utilized to evaluate and identify high molecular mass (>1000 Da) ellagitannins. The MALDI analysis verified the presence of the ellagitannins identified by HPLC-ESI-MS including lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D, but the analysis also indicated the presence of several other compounds that were most likely ellagitannins based on the patterns observed in the masses (i.e., loss or addition of a gallic acid moiety to a known ellagitannin). This study determined the presence of several possible isomeric forms of ellagitannins previously unidentified in fruit and presents a possible analytical HPLC method for the analysis of the major ellagitannins present in the fruit.

Quantitative HPLC Analysis of an Analgesic/Caffeine Formulation: Determination of Caffeine

NASA Astrophysics Data System (ADS)

Ferguson, Glenda K.

1998-04-01

A modern high performance liquid chromatography (HPLC) laboratory experiment which entails the separation of acetaminophen, aspirin, and caffeine and the quantitative assay of caffeine in commercial mixtures of these compounds has been developed. Our HPLC protocol resolves these compounds in only three minutes with a straightforward chromatographic apparatus which consists of a C-18 column, an isocratic mobile phase, UV detection at 254 nm, and an integrator; an expensive, sophisticated system is not required. The separation is both repeatable and rapid. Moreover, the experiment can be completed in a single three-hour period. The experiment is appropriate for any chemistry student who has completed a minimum of one year of general chemistry and is ideal for an analytical or instrumental analysis course. The experiment detailed herein involves the determination of caffeine in Goody's Extra Strength Headache Powders, a commercially available medication which contains acetaminophen, aspirin, and caffeine as active ingredients. However, the separation scheme is not limited to this brand of medication nor is it limited to caffeine as the analyte. With only minor procedural modifications, students can simultaneously quantitate all of these compounds in a commercial mixture. In our procedure, students prepare a series of four caffeine standard solutions as well as a solution from a pharmaceutical analgesic/caffeine mixture, chromatographically analyze each solution in quadruplicate, and plot relative average caffeine standard peak area versus concentration. From the mathematical relationship that results, the concentration of caffeine in the commercial formulation is obtained. Finally, the absolute standard deviation of the mean concentration is calculated.

Qualitative and quantitative analysis of the diuretic component ergone in Polyporus umbellatus by HPLC with fluorescence detection and HPLC-APCI-MS/MS.

PubMed

Zhao, Ying-Yong; Zhao, Ye; Zhang, Yong-Min; Lin, Rui-Chao; Sun, Wen-Ji

2009-06-01

Polyporus umbellatus is a widely used anti-aldosteronic diuretic in Traditional Chinese medicine (TCM). A new, sensitive and selective high-performance liquid chromatography-fluorescence detector (HPLC-FLD) and high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS/MS) method for quantitative and qualitative determination of ergosta-4,6,8(14),22-tetraen-3-one(ergone), which is the main diuretic component, was provided for quality control of P. umbellatus crude drug. The ergone in the ethanolic extract of P. umbellatus was unambiguously characterized by HPLC-APCI, and further confirmed by comparing with a standard compound. The trace ergone was detected by the sensitive and selective HPLC-FLD. Linearity (r2 > 0.9998) and recoveries of low, medium and high concentration (100.5%, 100.2% and 100.4%) were consistent with the experimental criteria. The limit of detection (LOD) of ergone was around 0.2 microg/mL. Our results indicated that the content of ergone in P. umbellatus varied significantly from habitat to habitat with contents ranging from 2.13 +/- 0.02 to 59.17 +/- 0.05 microg/g. Comparison among HPLC-FLD and HPLC-UV or HPLC-APCI-MS/MS demonstrated that the HPLC-FLD and HPLC-APCI-MS/MS methods gave similar quantitative results for the selected herb samples, the HPLC-UV methods gave lower quantitative results than HPLC-FLD and HPLC-APCI-MS/MS methods. The established new HPLC-FLD method has the advantages of being rapid, simple, selective and sensitive, and could be used for the routine analysis of P. umbellatus crude drug.

Simultaneous quantification of five steroid saponins from Dioscorea zingiberensis C.H.Wright in rat plasma by HPLC-MS/MS and its application to the pharmacokinetic studies

PubMed Central

Zhang, Xinxin; Li, Jing; Ito, Yoichiro; Sun, Wenji

2014-01-01

A simple, reliable and sensitive high-performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) was established for simultaneous analyses of the following 5 steroid saponins in rat plasma after the single dose administration of total steroid saponins extracted from the rhizome of Dioscorea zingiberensis C.H.Wright for the first time. Protodioscin, huangjiangsu A, zingiberensis new saponin, dioscin, and gracillin were quantified using ginsenoside Rb1 as the internal standard (IS). The plasma samples were pretreated by a single step acetonitrile-mediated protein precipitation. The chromatographic separation was performed on an Inersil ODS-3 C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase composed of acetonitrile and water containing 0.1% formic acid under a gradient elution mode at 0.2 mL min−1 using a microsplit after the eluent from the HPLC apparatus. The quantification was accomplished on a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) in the positive ionization mode. The above five analytes were stable under sample storage and preparation conditions applied in the present study. The linearity, precision, accuracy, and recoveries of the analysis confirmed the requirements for quality-control purposes. After validation, this proposed method was successfully adopted to investigate the pharmacokinetic parameters of these five analytes. PMID:25201262

Separation and preparation of xanthochymol and guttiferone E by high performance liquid chromatography and high speed counter-current chromatography combined with silver nitrate coordination reaction.

PubMed

Li, Jun; Gao, Ruixi; Zhao, Dan; Huang, Xianju; Chen, Yu; Gan, Fei; Liu, Hui; Yang, Guangzhong

2017-08-18

Xanthochymol (XCM) and guttiferone E (GFE), a pair of π bond benzophenone isomers from Garcinia xanthochymus, were once reported to be difficult or impossible to separate. The present study reports the successful separation of these two isomers through high performance liquid chromatography (HPLC), as well as their effective isolation using high speed counter-current chromatography (HSCCC) based on the silver nitrate (AgNO 3 ) coordination reaction. First, an effective HPLC separation system was developed, achieving a successful baseline separation with resolution of 2.0. Based on the partition coefficient (K) resolved by HPLC, the two-phase solvent system was determined as n-hexane, methanol and water with the uncommon volume ratio of 4:6:1. A crude extract of Garcinia xanthochymus (0.2g) was purified by normal HSCCC and refined with AgNO 3 -HSCCC. Monomers of XCM and GFE were identified by HPLC, mass spectrometry (MS) and nuclear magnetic resonance (NMR). The results demonstrate the separation and isolation of π bond benzophenone isomers using ordinary octadecyl silane (C 18 ) columns and HSCCC. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous separation and determination of fructose, sorbitol, glucose and sucrose in fruits by HPLC-ELSD.

PubMed

Ma, Chunmei; Sun, Zhen; Chen, Changbao; Zhang, Lili; Zhu, Shuhua

2014-02-15

A high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was optimised for simultaneous determination of fructose, sorbitol, glucose and sucrose in fruits. The analysis was carried out on a Phenomenex Luna 5u NH₂ 100A column (250 mm × 4.60mm, 5 micron) with isocratic elution of acetonitrile:water (82.5:17.5, v/v). Drift tube temperature of the ELSD system was set to 82 °C and nitrogen flow rate was 2.0 L min⁻¹. The regression equation revealed good linear relationship (R = 0.9967-0.9989) within test ranges. The limits of detection (LOD) and quantification (LOQ) for four analytes (peach, apple, watermelon, and cherry fruits) were in the range of 0.07-0.27 and 0.22-0.91 mg L⁻¹, respectively. The proposed HPLC-ELSD method was validated for quantification of sugars in peach, apple, watermelon, and cherry fruits, and the results were satisfactory. The results showed that the contents of the four sugars varied among fruits. While fructose (5.79-104.01 mg g⁻¹) and glucose (9.25-99.62 mg g⁻¹) emerged as common sugars in the four fruits, sorbitol (8.70-19.13 mg g⁻¹) were only found in peach, apple and cherry fruits, and sucrose (15.82-106.39 mg g⁻¹) were in peach, apple and watermelon. There was not detectable sorbitol in watermelon and sucrose in cherry fruits, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Correlation of HPLC Characteristic Spectra of Vinegar Corydalis Rhizoma Decoction Pieces, Water Decoction and Formula Granules].

PubMed

Wei, Mei; Du, Lan-zhe; Li, Hui; Zhang, Guang-da; Chen, Xiang-dong

2015-05-01

To study the correlation of characteristic spectra of Vinegar Corydalis Rhizoma decoction pieces, water decoction and formula granules by HPLC, and to investigate the transfer of the main chemical constituents between three different forms. The analysis was carried out by a Phenomenex Gemini C18 column (250 mm x 4.6 mm,5 μm) with acetonitrile-1% acetic acid and ammonium acetate buffer solution (pH 6.0) as the mobile phase in a gradient elution mode. The detection wavelength was 280 nm with a flow rate of 0.8 mL /min. The column temperature was 30 degrees C. The characteristic spectra from 11 batches of Vinegar Corydalis Rhizoma decoction pieces, 11 batches of water decoction and 11 batches of formula granules were established respectively. Ten peaks in the HPLC characteristic spectra from 11 batches of formula granules could be tracked in the water decoction, nine peaks in the HPLC characteristic spectra could be tracked in the decoction pieces. In the ten common peaks, four components such as protopine, palnatine chloride, berberine hydrochloride and tetrahydropalmatine were verified. The main chemical components of Vinegar Corydalis Rhizoma decoction pieces, water decoction and formula granules are basically the same, the common component contents have similar proportion.

Post-synthetic modification of MIL-101(Cr) with pyridine for high-performance liquid chromatographic separation of tocopherols.

PubMed

Yang, Fang; Yang, Cheng-Xiong; Yan, Xiu-Ping

2015-05-01

Effective separation of tocopherols is challenging and significant due to their structural similarity and important biological role. Here we report the post-synthetic modification of metal-organic framework (MOF) MIL-101(Cr) with pyridine for high-performance liquid chromatographic (HPLC) separation of tocopherols. Baseline separation of four tocopherols was achieved on a pyridine-grafted MIL-101(Cr) packed column within 10 min using hexane/isopropanol (96:4, v/v) as the mobile phase at a flow rate of 0.5 mL min(-1). The pyridine-grafted MIL-101(Cr) packed column gave high column efficiency (85,000 plates m(-1) for δ-tocopherol) and good precision (0.2-0.3% for retention time, 1.8-3.4% for peak area, 2.6-2.7% for peak height), and also offered much better performance than unmodified MIL-101(Cr) and commercial amino-bonded silica packed column for HPLC separation of tocopherols. The results not only show the promising application of pyridine-grafted MIL-101(Cr) as a novel stationary phase for HPLC separation of tocopherols, but also reveal a facile post-modification of MOFs to expand the application of MOFs in separation sciences. Copyright © 2015 Elsevier B.V. All rights reserved.

Performance analysis of a microfluidic mixer based on high gradient magnetic separation principles

NASA Astrophysics Data System (ADS)

Liu, Mengyu; Han, Xiaotao; Cao, Quanliang; Li, Liang

2017-09-01

To achieve a rapid mixing between a water-based ferrofluid and DI water in a microfluidic environment, a magnetically actuated mixing system based on high gradient magnetic separation principles is proposed in this work. The microfluidic system consists of a T-shaped mirochannel and an array of integrated soft-magnetic elements at the sidewall of the channel. With the aid of an external magnetic bias field, these elements are magnetized to produce a magnetic volume force acting on the fluids containing magnetic nanoparticles, and then to induce additional flows for improving the mixing performance. The mixing process is numerically investigated through analyzing the concentration distribution of magnetic nanoparticles using a coupled particle-fluid transport model, and mixing performances under different parametrical conditions are investigated in detail. Numerical results show that a high mixing efficiency around 97.5% can be achieved within 2 s under an inlet flow rate of 1 mm s-1 and a relatively low magnetic bias field of 50 mT. Meanwhile, it has been found that there is an optimum number of magnetic elements used for obtaining the best mixing performance. These results show the potential of the proposed mixing method in lab-on-a-chip system and could be helpful in designing and optimizing system performance.

Relationships of Quality Characteristics with Size Exclusion HPLC Chromatogram of Protein Extract in Soft-White Winter Wheats.

USDA-ARS?s Scientific Manuscript database

This study investigated relationships between molecular weight distributions of unreduced grain proteins and grain, flour, and end-use quality characteristics of soft white winter wheats grown in Oregon. Absorbance area and area % values of protein fractions separated by size exclusion HPLC (SE-HPL...

[Determination of biphenyl ether herbicides in water using HPLC with cloud-point extraction].

PubMed

He, Cheng-Yan; Li, Yuan-Qian; Wang, Shen-Jiao; Ouyang, Hua-Xue; Zheng, Bo

2010-01-01

To determine residues of multiple biphenyl ether herbicides simultaneously in water using high performance liquid chromatography (HPLC) with cloud-point extraction. The residues of eight biphenyl ether herbicides (including bentazone, fomesafen, acifluorfen, aclonifen, bifenox, fluoroglycofenethy, nitrofen, oxyfluorfen) in water samples were extracted with cloud-point extraction of Triton X-114. The analytes were separated and determined using reverse phase HPLC with ultraviolet detector at 300 nm. Optimized conditions for the pretreatment of water samples and the parameters of chromatographic separation applied. There was a good linear correlation between the concentration and the peak area of the analytes in the range of 0.05-2.00 mg/L (r = 0.9991-0.9998). Except bentazone, the spiked recoveries of the biphenyl ether herbicides in the water samples ranged from 80.1% to 100.9%, with relative standard deviations ranging from 2.70% to 6.40%. The detection limit of the method ranged from 0.10 microg/L to 0.50 microg/L. The proposed method is simple, rapid and sensitive, and can meet the requirements of determination of multiple biphenyl ether herbicides simultaneously in natural waters.

Performance optimization in electric field gradient focusing.

PubMed

Sun, Xuefei; Farnsworth, Paul B; Tolley, H Dennis; Warnick, Karl F; Woolley, Adam T; Lee, Milton L

2009-01-02

Electric field gradient focusing (EFGF) is a technique used to simultaneously separate and concentrate biomacromolecules, such as proteins, based on the opposing forces of an electric field gradient and a hydrodynamic flow. Recently, we reported EFGF devices fabricated completely from copolymers functionalized with poly(ethylene glycol), which display excellent resistance to protein adsorption. However, the previous devices did not provide the predicted linear electric field gradient and stable current. To improve performance, Tris-HCl buffer that was previously doped in the hydrogel was replaced with a phosphate buffer containing a salt (i.e., potassium chloride, KCl) with high mobility ions. The new devices exhibited stable current, good reproducibility, and a linear electric field distribution in agreement with the shaped gradient region design due to improved ion transport in the hydrogel. The field gradient was calculated based on theory to be approximately 5.76 V/cm(2) for R-phycoerythrin when the applied voltage was 500 V. The effect of EFGF separation channel dimensions was also investigated; a narrower focused band was achieved in a smaller diameter channel. The relationship between the bandwidth and channel diameter is consistent with theory. Three model proteins were resolved in an EFGF channel of this design. The improved device demonstrated 14,000-fold concentration of a protein sample (from 2 ng/mL to 27 microg/mL).

[Identification of chemical constituents in Sinopodophylli Fructus by HPLC-DAD-ESI-IT-TOF-MSn].

PubMed

Wang, Ai-Hua; Ma, Li-Man; Fan, Shan-Shan; Liu, Guang-Xue; Xu, Feng; Shang, Ming-Ying; Cai, Shao-Qing

2018-01-01

This experiment was performed to analyze and identify the chemical constituents of Sinopodophylli Fructus by HPLC-DAD-ESI-IT-TOF-MSn. The analysis was performed on an Agilent Zorbax SB-C₁₈ (4.6 mm×250 mm, 5 μm) column.The mobile phase consisted of 0.1% formic acid was used for gradient at a flow rate of 1.0 mL·min⁻¹. Electrospray ionization ion trap time-of-flight multistage mass spectrometry was applied for qualitative analysis under positive and negative ion modes. The results indicated that 54 compounds consisted of 18 lignans and 36 flavonoids from Xiaoyelian had been detected by their HRMS data, the information of literature and reference substance. Among them, 27 compounds were reported in Sinopodophylli Fructus for the first time. In conclusion, an HPLC-DAD-ESI-IT-TOF-MSn method was established to qualitative analysis of Xiaoyelian in this study, which will provide the evidence for evaluating the quality of Xiaoyelian herbs, clarifying the mechanism, and guiding the development of pharmacological active ingredients. Copyright© by the Chinese Pharmaceutical Association.

Separation of natural product using columns packed with Fused-Core particles.

PubMed

Yang, Peilin; Litwinski, George R; Pursch, Matthias; McCabe, Terry; Kuppannan, Krishna

2009-06-01

Three HPLC columns packed with 3 microm, sub-2 microm, and 2.7 microm Fused-Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express C18 column packed with Fused-Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3 C18 column packed with 3 microm particles. Column lot-to-lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity BEH column packed with sub-2 microm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high-efficiency and high-speed separation to be performed using conventional HPLC instrumentation.

Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC×LC-IT-TOF MS.

PubMed

Xu, Yu; Wang, DanDan; Tang, Lan; Wang, Jian

2017-10-25

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and characterized by a trap-free two-dimensional high performance size exclusion chromatography (HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion trap/time-of-flight mass spectrometry (2D-HPSEC×LC-IT-TOF MS) with positive and negative modes of electrospray ionization method. Separation and characterization the allergic polymerized impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8mm×30cm, 5μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second dimension analytical column was ZORBAX SB-C18 (4.6×150mm, 3.5μm) with ammonium formate solution (10mM) and ammonium formate (8mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase. Structures of eleven unknown impurities were deduced based on the high resolution MS n data with both positive and negative modes, in which nine impurities were polymerized impurities. The forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and online demineralization technique, which was worthy of widespread use and application for the advantages of stability and repeatability. Copyright © 2017. Published by Elsevier B.V.

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Furthermore, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

DOE PAGES

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

2016-04-07

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Furthermore, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

Improved HPLC method for the determination of curcumin, demethoxycurcumin, and bisdemethoxycurcumin.

PubMed

Jayaprakasha, Guddadarangavvanahally K; Jagan Mohan Rao, Lingamullu; Sakariah, Kunnumpurath K

2002-06-19

Commercially available curcumin, a bright orange-yellow color pigment of turmeric, consists of a mixture of three curcuminoids, namely, curcumin, demethoxycurcumin, and bisdemethoxycurcumin. These were isolated by column chromatography and identified by spectroscopic studies. The purity of the curcuminoids was analyzed by an improved HPLC method. HPLC separation was performed on a C(18) column using three solvents, methanol, 2% AcOH, and acetonitrile, with detection at 425 nm. Four different commercially available varieties of turmeric, namely, Salem, Erode, Balasore, and local market samples, were analyzed to detect the percentage of these three curcuminoids. The percentages of curcumin, demethoxycurcumin, and bisdemethoxycurcumin as estimated using their calibration curves were found to be 1.06 +/- 0.061 to 5.65 +/- 0.040, 0.83 +/- 0.047 to 3.36 +/- 0.040, and 0.42 +/- 0.036 to 2.16 +/- 0.06, respectively, in four different samples. The total percentages of curcuminoids are 2.34 +/- 0.171 to 9.18 +/- 0.232%.

Determination of arsenic species in marine samples by HPLC-ICP-MS.

PubMed

Hirata, Shizuko; Toshimitsu, Hideki; Aihara, Masato

2006-01-01

Arsenic speciation analysis in marine samples was performed using high performance liquid chromatography (HPLC) with ICP-MS detection. The separation of eight arsenic species viz. arsenite (As(III)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenate (As(V)), arsenobetaine, trimethylarsine oxide (TMAO), arsenocholine and tetramethylarsonium ion (TeMAs) was achieved on a Shiseido Capcell Pak C18 column by using an isocratic eluent (pH 3.0), in which condition As(III) and MMA were co-eluted. The entire separation was accomplished in 15 min. The detection limits for 8 arsenic species by HPLC/ICP-MS were in the range of 0.02 - 0.10 microg L(-1) based on 3sigma of blank response (n=9). The precision was calculated to be 3.1-7.3% (RSD) for all eight species. The method then successfully applied to several marine samples e.g., oyster, scallop, fish, and shrimps. For the extraction of arsenic species from seafood products, the low power microwave digestion was employed. The extraction efficiency was in the range of 52.9 - 112.3%. Total arsenic concentrations were analyzed by using the microwave acid digestion. The total arsenics in the certified reference materials (DORM-2 and TORT-2) were analyzed and agreed with the certified values. The concentrations of arsenics in marine samples were in the range 6.6 - 35.1 microg g(-1).

HPLC: Early and Recent Perspectives.

ERIC Educational Resources Information Center

Karger, Barry L.

1997-01-01

Provides a perspective on what it was like in the early days of high-performance liquid chromatography (HPLC) and several of the key developments. Focuses on the advances in HPLC generally, and more specifically for the biological sciences, that were necessary for the method to reach the preeminent stage of today. Contains 20 references. (JRH)

DryLab® optimised two-dimensional high performance liquid chromatography for differentiation of ephedrine and pseudoephedrine based methamphetamine samples.

PubMed

Andrighetto, Luke M; Stevenson, Paul G; Pearson, James R; Henderson, Luke C; Conlan, Xavier A

2014-11-01

In-silico optimised two-dimensional high performance liquid chromatographic (2D-HPLC) separations of a model methamphetamine seizure sample are described, where an excellent match between simulated and real separations was observed. Targeted separation of model compounds was completed with significantly reduced method development time. This separation was completed in the heart-cutting mode of 2D-HPLC where C18 columns were used in both dimensions taking advantage of the selectivity difference of methanol and acetonitrile as the mobile phases. This method development protocol is most significant when optimising the separation of chemically similar chemical compounds as it eliminates potentially hours of trial and error injections to identify the optimised experimental conditions. After only four screening injections the gradient profile for both 2D-HPLC dimensions could be optimised via simulations, ensuring the baseline resolution of diastereomers (ephedrine and pseudoephedrine) in 9.7 min. Depending on which diastereomer is present the potential synthetic pathway can be categorized.

Comparative HPLC/ESI-MS and HPLC/DAD study of different populations of cultivated, wild and commercial Gentiana lutea L.

PubMed

Mustafa, Ahmed M; Caprioli, Giovanni; Ricciutelli, Massimo; Maggi, Filippo; Marín, Rosa; Vittori, Sauro; Sagratini, Gianni

2015-05-01

The root of Gentiana lutea L., famous for its bitter properties, is often used in alcoholic bitter beverages, food products and traditional medicine to stimulate the appetite and improve digestion. This study presents a new, fast, and accurate HPLC method using HPLC/ESI-MS and HPLC/DAD for simultaneous analysis of iridoids (loganic acid), secoiridoids (gentiopicroside, sweroside, swertiamarin, amarogentin) and xanthones (isogentisin) in different populations of G.lutea L., cultivated in the Monti Sibillini National Park, obtained wild there, or purchased commercially. Comparison of HPLC/ESI-MS and HPLC/DAD indicated that HPLC/ESI-MS is more sensitive, reliable and selective. Analysis of twenty samples showed that gentiopicroside is the most dominant compound (1.85-3.97%), followed by loganic acid (0.11-1.30%), isogentisin (0.03-0.48%), sweroside (0.05-0.35%), swertiamarin (0.08-0.30%), and amarogentin (0.01-0.07%). The results confirmed the high quality of the G.lutea cultivated in the Monti Sibillini National Park. Copyright © 2014 Elsevier Ltd. All rights reserved.

Flow separation of currents in shallow water

USGS Publications Warehouse

Signell, Richard P.

1989-01-01

Flow separation of currents in shallow coastal areas is investigated using a boundary layer model for two-dimensional (depth-averaged) tidal flow past an elliptic headland. If the shoaling region near the coast is narrow compared to the scale of the headland, bottom friction causes the flow to separate just downstream of the point where the pressure gradient switches from favoring to adverse. As long as the shoaling region at the coast is well resolved, the inclusion of eddy viscosity and a no-slip boundary condition have no effect on this result. An approximate analytic solution for the pressure gradient along the boundary is obtained by assuming the flow away from the immediate vicinity of the boundary is irrotational. On the basis of the pressure gradient obtained from the irrotational flow solution, flow separation is a strong function of the headland aspect ratio, an equivalent Reynolds number, and a Keulegan-Carpenter number.

HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp.

PubMed

Varik, Vallo; Oliveira, Sofia Raquel Alves; Hauryliuk, Vasili; Tenson, Tanel

2017-09-08

Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved experiments feasible. The method combines cell harvesting by rapid filtration, followed by acid extraction, freeze-drying with chromatographic separation. We use a combination of C18 IPRP-HPLC (GMP unresolved and co-migrating with IMP; GDP and GTP; AMP, ADP and ATP; CTP; UTP) and SAX-HPLC in isocratic mode (ppGpp and pppGpp) with UV detection. The approach is applicable to bacteria without the requirement of metabolic labelling with 32P-labelled radioactive precursors. We applied our method to quantify nucleotide pools in Escherichia coli BW25113 K12-strain both throughout the growth curve and during acute stringent response induced by mupirocin. While ppGpp and pppGpp levels vary drastically (40- and ≥8-fold, respectively) these changes are decoupled from the quotients of the housekeeping pool and guanosine and adenosine housekeeping nucleotides: NTP/NDP/NMP ratio remains stable at 6/1/0.3 during both normal batch culture growth and upon acute amino acid starvation.

Reynolds stress structures in a self-similar adverse pressure gradient turbulent boundary layer at the verge of separation.

NASA Astrophysics Data System (ADS)

Atkinson, C.; Sekimoto, A.; Jiménez, J.; Soria, J.

2018-04-01

Mean Reynolds stress profiles and instantaneous Reynolds stress structures are investigated in a self-similar adverse pressure gradient turbulent boundary layer (APG-TBL) at the verge of separation using data from direct numerical simulations. The use of a self-similar APG-TBL provides a flow domain in which the flow gradually approaches a constant non-dimensional pressure gradient, resulting in a flow in which the relative contribution of each term in the governing equations is independent of streamwise position over a domain larger than two boundary layer thickness. This allows the flow structures to undergo a development that is less dependent on the upstream flow history when compared to more rapidly decelerated boundary layers. This APG-TBL maintains an almost constant shape factor of H = 2.3 to 2.35 over a momentum thickness based Reynolds number range of Re δ 2 = 8420 to 12400. In the APG-TBL the production of turbulent kinetic energy is still mostly due to the correlation of streamwise and wall-normal fluctuations, 〈uv〉, however the contribution form the other components of the Reynolds stress tensor are no longer negligible. Statistical properties associated with the scale and location of sweeps and ejections in this APG-TBL are compared with those of a zero pressure gradient turbulent boundary layer developing from the same inlet profile, resulting in momentum thickness based range of Re δ 2 = 3400 to 3770. In the APG-TBL the peak in both the mean Reynolds stress and the production of turbulent kinetic energy move from the near wall region out to a point consistent with the displacement thickness height. This is associated with a narrower distribution of the Reynolds stress and a 1.6 times higher relative number of wall-detached negative uv structures. These structures occupy 5 times less of the boundary layer volume and show a similar reduction in their streamwise extent with respect to the boundary layer thickness. A significantly lower percentage

High-speed and high-resolution UPLC separation at zero degrees Celsius

PubMed Central

Wales, Thomas E.; Fadgen, Keith E.; Gerhardt, Geoff C.; Engen, John R.

2008-01-01

The conformational properties of proteins can be probed with hydrogen/deuterium exchange mass spectrometry (HXMS). In order to maintain the deuterium label during LC/MS analyses, chromatographic separation must be done rapidly (usually in under 8–10 minutes) and at zero degrees Celsius. Traditional RP-HPLC with ~3 micron particles has shown generally poor chromatographic performance under these conditions and thereby has been prohibitive for HXMS analyses of larger proteins and many protein complexes. Ultra performance liquid chromatography (UPLC) employs particles smaller than 2 microns in diameter to achieve superior resolution, speed, and sensitivity as compared to HPLC. UPLC has previously been shown to be compatible with the fast separation and low temperature requirements of HXMS. Here we present construction and validation of a custom UPLC system for HXMS. The system is based on the Waters nanoACQUITY platform and contains a Peltier-cooled module that houses the injection and switching valves, online pepsin digestion column, and C-18 analytical separation column. Single proteins in excess of 95 kDa and a four-protein mixture in excess of 250 kDa have been used to validate the performance of this new system. Near baseline resolution was achieved in 6 minute separations at 0 °C and displayed a median chromatographic peak width of ~2.7 sec at half height. Deuterium recovery was similar to that obtained using a conventional HPLC and icebath. This new system represents a significant advancement in HXMS technology that is expected to make the technique more accessible and mainstream in the near future. PMID:18672890

Study of rat hypothalamic proteome by HPLC/ESI ion trap and HPLC/ESI-Q-TOF MS.

PubMed

Iqbal, Javed; Li, Wang; Ullah, Kaleem; Hasan, Murtaza; Linna, Guo; Awan, Umer; Zhang, Yongqian; Batool, Sajida; Qing, Hong; Deng, Yulin

2013-08-01

The proteomic profile of hypothalamus, a key organ of CNS, is explored here by employing two widely used MS techniques, i.e. HPLC/ESI-ion trap and HPLC/ESI-quadrupole-TOF MS. Strong cation exchange is used for the fractionation of peptides and protein search engine MASCOT is employed for data query. One hundred and thirty six proteins with 10 973 peptides were identified by HPLC/ESI-ion trap MS, while 140 proteins with 32 183 peptides were characterized by HPLC/ESI-quadrupole-TOF MS. Among the total 198 proteins identified in both experiments, 78 proteins were common in both sets of conditions. The rest of the 120 proteins were identified distinctly in both MS strategies, i.e. 58 unique proteins were found using the quadrupole-TOF while 62 were found with the HPLC/ESI-ion trap. Moreover, these proteins were classified into groups based on their functions performed in the body. Results presented here identified some important signal and cellular defense proteins inevitable for survival in stressed conditions. Additionally, it is also shown that any single MS strategy is not reliable for good results due to loss of data depending on sensitivity of the instrument used. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of ionic chloroacetanilide-herbicide metabolites in surface water and groundwater by HPLC/MS using negative ion spray

USGS Publications Warehouse

Ferrer, I.; Thurman, E.M.; Barcelo, D.

1997-01-01

Solid-phase extraction (SPE) was combined with high-performance liquid chromatography/high-flow pneumatically assisted electrospray mass spectrometry (HPLC/ESP/MS) for the trace analysis of oxanilic and sulfonic acids of acetochlor, alachlor, and metolachlor. The isolation procedure separated the chloroacetanilide metabolites from the parent herbicides during the elution from C18 cartridges using ethyl acetate for parent compounds, followed by methanol for the anionic metabolites. The metabolites were separated chromatographically using reversed-phase HPLC and analyzed by negative-ion MS using electrospray ionization in selected ion mode. Quantitation limits were 0.01 ??g/L for both the oxanilic and sulfonic acids based on a 100-mL water sample. This combination of methods represents an important advance in environmental analysis of chloroacetanilide-herbicide metabolites in surface water and groundwater for two reasons. First, anionic chloroacetanilide metabolites are a major class of degradation products that are readily leached to groundwater in agricultural areas. Second, anionic metabolites, which are not able to be analyzed by conventional methods such as liquid extraction and gas chromatography/mass spectrometry, are effectively analyzed by SPE and high-flow pneumatically assisted electrospray mass spectrometry. This paper reports the first HPLC/MS identification of these metabolites in surface water and groundwater.

Improved method for reliable HMW-GS identification by RP-HPLC and SDS-PAGE in common wheat cultivars

USDA-ARS?s Scientific Manuscript database

The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differe...

Identification and structural characterisation of triterpene saponins from the root of Ardisia mamillata Hance by HPLC-ESI-QTOF-MS/MS.

PubMed

Zhang, Er-Fei; Ling, Yun; Yin, Zi; Zhang, Qing

2018-04-01

Triterpene saponins in medicinal plants attract scientific attentions for their structural diversity and significant bioactivities. In this work, a high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS) method is used to rapidly separate and identify triterpene saponins from the extract of Ardisia mamillata Hance (AMH). In the full scan mass spectrum, the accurate determination of molecular formula is obtained by the predominant ion [M + HCOO] - in negative ion mode. As a result, 30 triterpene saponins are identified or tentatively identified in the plant extract. Of these, 17 triterpene saponins are new compounds. In conclusion, the HPLC-ESI-QTOF-MS/MS is an efficient technique to separate and identify triterpene saponins in complex matrices of medicinal plant.

A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma

PubMed Central

Zhu, Hao-Jie; Wang, Jun-Sheng; Patrick, Kennerly S.; Donovan, Jennifer L.; DeVane, C. Lindsay; Markowitz, John S.

2007-01-01

A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (λex: 330 nm, λem: 460 nm). The lower limit of determination was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 to 80 ng/mL (r=0.998). The relative standard deviations of intra-day and inter-day variations were ≤ 9.10 % and ≤ 7.58 %, respectively. The accuracy ranged between 92.59 % and 103.06 %. The method was successfully applied to the pharmacokinetic study of a subject who received a single oral dose (0.3 mg/kg) of immediate-release MPH and yielded consistent results with that of the GC-MS method. This method is the first HPLC assay with non-MS detection providing sufficient reliability and sensitivity for both pre-clinical and clinical studies of MPH. PMID:17804308

Features of separating turbulent boundary layers

NASA Technical Reports Server (NTRS)

Nagabushana, K. A.; Agarwal, Naval K.; Simpson, Roger L.

1988-01-01

In the present study of two strong adverse pressure gradient flows, mean flow and turbulence characteristics are measured, together with frequency spectra, using hot-wire and laser anemometry. In these separating flows, reattachment occurs over a shorter distance than separation. It is noted that the outer flow variables form a unique set of scaling parameters for streamwise power spectra in adverse pressure gradient flows. The inner flow scaling of Perry et al. (1985) for streamwise spectra does not hold in the backflow region unless the value of the downstream-upstream intermittency in the flow is unity.

Influence of permittivity on gradient force exerted on Mie spheres.

PubMed

Chen, Jun; Li, Kaikai; Li, Xiao

2018-04-01

In optical trapping, whether a particle could be stably trapped into the focus region greatly depends on the strength of the gradient force. Individual theoretical study on gradient force exerted on a Mie particle is rare because the mathematical separation of the gradient force and the scattering force in the Mie regime is difficult. Based on the recent forces separation work by Du et al. [Sci. Rep.7, 18042 (2017)SRCEC32045-232210.1038/s41598-017-17874-1], we investigate the influence of permittivity (an important macroscopic physical quantity) on the gradient force exerted on a Mie particle by cooperating numerical calculation using fast Fourier transform and analytical analysis using multipole expansion. It is revealed that gradient forces exerted on small spheres are mainly determined by the electric dipole moment except for certain permittivity with which the real part of polarizability of the electric dipole approaches zero, and gradient forces exerted on larger spheres are complex because of the superposition of the multipole moments. The classification of permittivity corresponding to different varying tendencies of gradient forces exerted on small spheres or larger Mie particles are illustrated. Absorption of particles favors the trapping of small spheres by gradient force, while it is bad for the trapping of larger particles. Moreover, the absolute values of the maximal gradient forces exerted on larger Mie particles decline greatly versus the varied imaginary part of permittivity. This work provides elaborate investigation on the different varying tendencies of gradient forces versus permittivity, which favors more accurate and free optical trapping.

Assessing the scalability of dynamic field gradient focusing by linear modeling

PubMed Central

Tracy, Noah I.; Ivory, Cornelius F.

2010-01-01

Dynamic field gradient focusing (DFGF) separates and concentrates proteins in native buffers, where proteins are most soluble, using a computer-controlled electric field gradient which lets the operator adjust the pace and resolution of the separation in real-time. The work in this paper assessed whether DFGF could be scaled up from microgram analytical-scale protein loads to milligram preparative-scale loads. Linear modeling of the electric potential, protein transport, and heat transfer simulated the performance of a preparative-scale DFGF instrument. The electric potential model showed where the electrodes should be placed to optimize the shape and strength of the electric field gradient. Results from the protein transport model suggested that in 10 min the device should separate 10 mg each of two proteins whose electrophoretic mobilities differ by 5 ×. Proteins with electrophoretic mobilities differing by only 5% should separate in 3 h. The heat transfer model showed that the preparative DFGF design could dissipate 1 kW of Joule heat while keeping the separation chamber at 25°C. Model results pointed to DFGF successfully scaling up by 1000 × using the proposed instrument design. PMID:18196522

A HPLC-UV method for the determination of puerarin in rat plasma after intravenous administration of PEGylated puerarin conjugate.

PubMed

Liu, Xinyi; Zhi, Hongying; Du, Feng; Ye, Zuguang; Wang, Naijie; Qin, Wenjie; Li, Jianrong

2010-12-01

A sensitive and reproducible HPLC method for quantitative determination of puerarin (PUE) in rat plasma was developed and validated using 4-hydroxybenzaldehyde as an internal standard. The separation of PUE was performed on a CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The method was validated and found to be linear in the range of 80-12,000ng/mL. The limit of quantification was 80ng/mL based on 100μL of plasma. The variations for intra- and inter-day precision were less than 8.3%, and the accuracy values were between 98% and 105.2%. The extraction recoveries were more than 85%. The method was successfully applied in the comparative study of pharmacokinetics of PEGylated puerarin (PEG-PUE) versus PUE in rats. Compared with PUE, PEG-PUE showed a 5.2-fold increase in half-life of PUE and a 4.7-fold increase in mean residence time. In addition, this method was also successfully applied to determine the low plasma concentration of PUE regenerated from PEG-PUE in vitro. Copyright © 2010 Elsevier B.V. All rights reserved.

Density gradients at hydrogel interfaces for enhanced cell penetration.

PubMed

Simona, B R; Hirt, L; Demkó, L; Zambelli, T; Vörös, J; Ehrbar, M; Milleret, V

2015-04-01

We report that stiffness gradients facilitate infiltration of cells through otherwise cell-impermeable hydrogel interfaces. By enabling the separation of hydrogel manufacturing and cell seeding, and by improving cell colonization of additively manufactured hydrogel elements, interfacial density gradients present a promising strategy to progress in the creation of 3D tissue models.

HPLC Analysis of Chlorophyll a, Chlorophyll b, and Beta-Carotene in Collard Greens: A Project for a Problem-Oriented Laboratory Course.

ERIC Educational Resources Information Center

Silveira, Augustine, Jr.; And Others

1984-01-01

High performance liquid chromatography (HPLC) is used to separate and quantitate beta-carotene, chlorophyll a, and chlorophyll b originating from collard greens. Experimental procedures used and typical results obtained are discussed. (JN)

A high gradient and strength bioseparator with nano-sized immunomagnetic particles for specific separation and efficient concentration of E. coli O157:H7

NASA Astrophysics Data System (ADS)

Lin, Jianhan; Li, Min; Li, Yanbin; Chen, Qi

2015-03-01

Sample pretreatment is a key to rapid screening of pathogens for prevention and control of foodborne diseases. Magnetic immunoseparation is a specific method based on antibody-antigen reaction to capture the target bacteria and concentrate them in a smaller-volume buffer. The use of nano-sized magnetic particles could improve the separation efficiency of bacteria but require much higher gradient and strength magnetic field. In this study, a strong magnetic bioseparator with a mean field strength of 1.35 T and a mean gradient of 90 T/m was developed with the use of the 30 nm and 180 nm magnetic particles to specifically separate and efficiently concentrate foodborne bacterial pathogens using Escherichia coli O157:H7 as a model bacterium. The polyclonal antibodies against E. coli were evaluated using Dot ELISA analysis for their good affinity with the target bacteria and then used to modify the surface of the magnetic nanoparticles by 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) method and streptavidin-biotin binding. The magnetic particle concentrations were optimized to be 40 μg/ml and 100 μg/ml for the 30 nm and 180 nm particles, respectively, the immunoreaction time was optimized to be 45 min for both sizes of particles, and the separation times were optimized to be 60 min and 2 min for the 30 nm and 180 nm particles, respectively. The total magnetic separation time was 2 h and 1 h for the 30 nm and 180 nm particles, respectively. The experimental results demonstrated that the bioseparator with the use of either 30 nm or 180 nm immunomagnetic particles could achieve a separation efficiency of >90% for E. coli O157:H7 at the concentrations ranging from 102 to 105 cfu/ml. No obvious interferences from non-target foodborne pathogens, such as SalmonellaTyphimurium and Listeria innocua, were found. For overall consideration of the consuming time, the cost, and the separation efficiency, the 180 nm magnetic particles are practical for rapid

Determination and Isolation of Four Anti-tumour Saponins from Lonicera macranthoides by HPLC-ESI-QTOF/MS and HSCCC.

PubMed

Liu, Xuehui; Zhou, Rongrong; Shen, Bingbing; Chen, Lin; Zhou, Qingyijun; Wan, Dan; Huang, Luqi; Zhang, Shuihan

2017-01-01

Lonicera macranthoides is a Chinese herb that contains a large number of bioactive spanions possessing important pharmacological activities, such as anti-tumour activity. However, detailed information about their anti-tumor activity and bioactive compounds is limited. In order to evaluate the scientific basis, the method of high-speed counter-current chromatography (HSCCC) combined with high performance liquid chromatography mass spectrometry (HPLC-ESI-QTOF/MS) has been developed to separate, purify and analyze saponins from Lonicera macranthoides. Four main saponins, Macranthoidin B (I), Macranthoidin A (II), Macranthoides B (III) and Akebia saponin D (IV) were separated by HSCCC with the solvent systems of ethyl acetate-nbutanol- water (3:2:5) and n-butanol-water-methanol-ethyl acetate (1:6:0.5:4). The purities of these four bioactive ingredients (I-IV) identified and detected by HPLC-ESI-QTOF/MS were 95.1%, 92.7%, 91.8% and 96.3%, respectively. The separated saponins were evaluated for their cytotoxic activities against six tumor cell lines (MCF-7, Hela, A549, HepG2, HT29 and Eca109). Results show that compounds I-IV exhibited particular significant anti-tumor activities against human mammary adenocarcinoma MCF-7 cell with IC50 values ranging from 12.7 to 30.8 µM. It was demonstrated that the combinative method using HPLC-ESI-QTOF/MS and HSCCC was suitable for rapid screening and isolating saponins of Lonicera macranthoides and the isolated compounds have great potential for the development of new antitumor drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Closed-loop Separation Control Using Oscillatory Flow Excitation

NASA Technical Reports Server (NTRS)

Allan, Brian G.; Juang, Jer-Nan; Raney, David L.; Seifert, Avi; Pack, latunia G.; Brown, Donald E.

2000-01-01

Design and implementation of a digital feedback controller for a flow control experiment was performed. The experiment was conducted in a cryogenic pressurized wind tunnel on a generic separated configuration at a chord Reynolds number of 16 million and a Mach number of 0.25. The model simulates the upper surface of a 20% thick airfoil at zero angle-of-attack. A moderate favorable pressure gradient, up to 55% of the chord, is followed by a severe adverse pressure gradient which is relaxed towards the trailing edge. The turbulent separation bubble, behind the adverse pressure gradient, is then reduced by introducing oscillatory flow excitation just upstream of the point of flow separation. The degree of reduction in the separation region can be controlled by the amplitude of the oscillatory excitation. A feedback controller was designed to track a given trajectory for the desired degree of flow reattachment and to improve the transient behavior of the flow system. Closed-loop experiments demonstrated that the feedback controller was able to track step input commands and improve the transient behavior of the open-loop response.

Simultaneous Quantitative Determination of 12 Active Components in Yuanhu Zhitong Prescription by RP-HPLC Coupled with Photodiode Array Detection

PubMed Central

Zhang, Xiaokai; Zhang, Song; Yang, Qian; Cao, Wei; Xie, Yanhua; Qiu, Pengcheng; Wang, Siwang

2015-01-01

Background: Yuanhu Zhitong prescription (YZP) is a famous traditional Chinese medicine formula, which is officially recorded in Chinese Pharmacopoeia for the treatment of stomach pain, hypochondriac pain, headache and dysmenorrhea caused by qi-stagnancy and blood stasis. It is the first report for the simultaneous determination of 12 active components in YZP. Objective: A newly, simple, accurate and reliable method for the separation and determination of 12 active components (protopine, α-allocryptopine, coptisine, xanthotol, palmatine, dehydrocorydaline, glaucine, tetrahydropalmatine, tetrahydroberberine, imperatorin, corydaline, isoimperatorin) in YZP was developed and validated using HPLC-PAD. Materials and Methods: The analytes were performed on a Phenomenex Luna-C18 (2) column (250×4.6 mm, 5.0 μm) with a gradient elution program using a mixture of acetonitrile and 0.1% phosphoric acid water solution (adjusted with triethylamine to pH 5.6) as mobile phase. Analytes were performed at 30°C with a flow rate of 1.0 mL/min. Results: The validated method was applied to analyze four major dosage forms of YZP coming from different manufacturers with good linearity (r2, 0.9981~0.9999), precision (RSD, 0.24~2.89%), repeatability (RSD, 0.15~3.34%), stability (RSD, 0.14~3.35%), recovery (91.13~110.81%) of the 12 components. Conclusion: The proposed method enables the separation and determination of 12 active components in a single run for the quality control of YZP. PMID:25709212

Miniaturized protein separation using a liquid chromatography column on a flexible substrate

NASA Astrophysics Data System (ADS)

Yang, Yongmo; Chae, Junseok

2008-12-01

We report a prototype protein separator that successfully miniaturizes existing technology for potential use in biocompatible health monitoring implants. The prototype is a liquid chromatography (LC) column (LC mini-column) fabricated on an inexpensive, flexible, biocompatible polydimethylsiloxane (PDMS) enclosure. The LC mini-column separates a mixture of proteins using size exclusion chromatography (SEC) with polydivinylbenzene beads (5-20 µm in diameter with 10 nm pore size). The LC mini-column is smaller than any commercially available LC column by a factor of ~11 000 and successfully separates denatured and native protein mixtures at ~71 psi of the applied fluidic pressure. Separated proteins are analyzed using NuPAGE-gel electrophoresis, high-performance liquid chromatography (HPLC) and an automated electrophoresis system. Quantitative HPLC results demonstrate successful separation based on intensity change: within 12 min, the intensity between large and small protein peaks changed by a factor of ~20. In further evaluation using the automated electrophoresis system, the plate height of the LC mini-column is between 36 µm and 100 µm. The prototype LC mini-column shows the potential for real-time health monitoring in applications that require inexpensive, flexible implant technology that can function effectively under non-laboratory conditions.

DETERMINATION OF VENLAFAXINE, VILAZODONE AND THEIR MAIN ACTIVE METABOLITES IN HUMAN SERUM BY HPLC-DAD AND HPLC-MS.

PubMed

Petruczynik, Anna; Wroblewski, Karol; Szultka-Mlynska, Malgorzata; Buszewsk, Boguslaw; Karakula-Juchnowicz, Hanna; Gajewski, Jacek; Morylowska-Topolska, Justyna; Waksmundzka-Hajnosi, Monika

2017-05-01

A high performance liquid chromatography (HPLC) method for simultaneous analysis of venlafaxine and its major metabolite 0-desmethylvenlafaxine and vilazodone and its methabolite M10 have been devel- oped and validated. Chromatography was performed on the Phenyl-Hexyl column with mobile phase containing methanol, acetate buffer at pH 3.5 and diethylamine. The application of stationary phase with 7r-7c moieties and mobile phase containing diethylamine as silanol blocker lets to obtain double protection against silanols and thus very high theoretical plate numbers were obtained. The good separation selectivity, good peaks' symmetry and very high systems efficiency for all investigated compounds were obtained in applied chromatographic system. The method is very efficient and suitable for the analysis of investigated drugs and their metabolites in human serum for patients' pharmacotherapy control.

An Experiment Using Sucrose Density Gradients in the Undergraduate Biochemistry Laboratory.

ERIC Educational Resources Information Center

Turchi, Sandra L.; Weiss, Monica

1988-01-01

Describes an experiment to be performed in an undergraduate biochemistry laboratory that is based on a gradient centrifugation system employing a simple bench top centrifuge, a freezer, and frozen surcose gradient solution to separate macromolecules and subcellular components. (CW)

Texas A&M vortex type phase separator

NASA Astrophysics Data System (ADS)

Best, Frederick

2000-01-01

Phase separation is required for regenerative biological and chemical process systems as well as thermal transport and rejection systems. Liquid and gas management requirements for future spacecraft will demand small, passive systems able to operate over wide ranges of inlet qualities. Conservation and recycling of air and water is a necessary part of the construction and operation of the International Space Station as well as future long duration space missions. Space systems are sensitive to volume, mass, and power. Therefore, it is necessary to develop a method to recycle wastewater with minimal power consumption. Regenerative life support systems currently being investigated require phase separation to separate the liquid from the gas produced. The microgravity phase separator designed and fabricated at Texas A&M University relies on centripetal driven buoyancy forces to form a gas-liquid vortex within a fixed, right-circular cylinder. Two-phase flow is injected tangentially along the inner wall of this cylinder producing a radial acceleration gradient. The gradient produced from the intrinsic momentum of the injected mixture results in a rotating flow that drives the buoyancy process by the production of a hydrostatic pressure gradient. Texas A&M has flown several KC-135 flights with separator. These flights have included scaling studies, stability and transient investigations, and tests for inventory instrumentation. Among the hardware tested have been passive devices for separating mixed vapor/liquid streams into single-phase streams of vapor only and liquid only. .

Comparing monolithic and fused core HPLC columns for fast chromatographic analysis of fat-soluble vitamins.

PubMed

Kurdi, Said El; Muaileq, Dina Abu; Alhazmi, Hassan A; Bratty, Mohammed Al; Deeb, Sami El

2017-06-27

HPLC stationary phases of monolithic and fused core type can be used to achieve fast chromatographic separation as an alternative to UPLC. In this study, monolithic and fused core stationary phases are compared for fast separation of four fat-soluble vitamins. Three new methods on the first and second generation monolithic silica RP-18e columns and a fused core pentafluoro-phenyl propyl column were developed. Application of three fused core columns offered comparable separations of retinyl palmitate, DL-α-tocopheryl acetate, cholecalciferol and menadione in terms of elution speed and separation efficiency. Separation was achieved in approx. 5 min with good resolution (Rs > 5) and precision (RSD ≤ 0.6 %). Monolithic columns showed, however, a higher number of theoretical plates, better precision and lower column backpressure than the fused core column. The three developed methods were successfully applied to separate and quantitate fat-soluble vitamins in commercial products.

Simultaneous determination of bioactive compounds in Piper nigrum L. and a species comparison study using HPLC-PDA.

PubMed

Rao, Vidadala Rama Subba; Raju, Sagi Satyanarayana; Sarma, Vanka Umamaheswara; Sabine, Fouriner; Babu, Kothapalli Hari; Babu, Katragadda Suresh; Rao, Janaswamy Madhusudana

2011-08-01

Piper nigrum L. is a traditional medicine widely used in India for illnesses such as constipation, diarrhoea, earache, gangrene, heart disease, hernia, hoarseness, indigestion, insect bites, insomnia, joint pain, liver problems, lung disease, oral abscesses, sunburn, tooth decay and toothaches. In this study, six bioactive compounds, namely piperine (1), pellitorine (2), guineensine (3), pipnoohine (4), trichostachine (5) and piperonal (6) were quantified in different extracts of P. nigrum L. and compared with those of P. longum L. and P. chaba Hunter. To evaluate the quality of P. nigrum, a simple, accurate and precise HPLC-PDA method was developed for the simultaneous determination of the above-mentioned six compounds. The separation was achieved by Phenomenex Luna RP C(18) column (150 × 4.6 mm, 5 µm, Phenomenex Inc, CA, USA) with a binary gradient solvent system of water-acetonitrile, at a flow rate of 1.0 mL min(-1) and detected at 210, 232, 262 and 343 nm. All six calibration curves showed good linearity (R (2) > 0.9966). The method was reproducible with intra- and inter-day variations of less than 2% and 5%, respectively. The results demonstrated that this method is simple, reliable and suitable for the quality control of these plants.

Technical Note: Interleaved Bipolar Acquisition and Low-rank Reconstruction for Water-Fat Separation in MRI.

PubMed

Cho, JaeJin; Park, HyunWook

2018-05-17

To acquire interleaved bipolar data and reconstruct the full data using low-rank property for water fat separation. Bipolar acquisition suffers from issues related to gradient switching, the opposite gradient polarities, and other system imperfections, which prevent accurate water-fat separation. In this study, an interleaved bipolar acquisition scheme and a low-rank reconstruction method were proposed to reduce issues from the bipolar gradients while achieving a short imaging time. The proposed interleaved bipolar acquisition scheme collects echo-time signals from both gradient polarities; however, the sequence increases the imaging time. To reduce the imaging time, the signals were subsampled at every dimension of k-space. The low-rank property of the bipolar acquisition was defined and exploited to estimate the full data from the acquired subsampled data. To eliminate the bipolar issues, in the proposed method, the water-fat separation was performed separately for each gradient polarity, and the results for the positive and negative gradient polarities were combined after the water-fat separation. A phantom study and in-vivo experiments were conducted on a 3T Siemens Verio system. The results for the proposed method were compared with the results of the fully sampled interleaved bipolar acquisition and Soliman's method, which was the previous water-fat separation approach for reducing the issues of bipolar gradients and accelerating the interleaved bipolar acquisition. The proposed method provided accurate water and fat images without the issues of bipolar gradients and demonstrated a better performance compared with the results of the previous methods. The water-fat separation using the bipolar acquisition has several benefits including a short echo-spacing time. However, it suffers from bipolar-gradient issues such as strong gradient switching, system imperfection, and eddy current effects. This study demonstrated that accurate water-fat separated images can

Density gradient centrifugation: Application to the separation of macerals of type I, II, and III sedimentary organic matter

USGS Publications Warehouse

Stankiewicz, B.A.; Kruge, M.A.; Crelling, J.C.; Salmon, G.L.

1994-01-01

Samples of organic matter from nine well-known geological units (Green River Fm., Tasmanian Tasmanite, Lower Toarcian Sh. of the Paris Basin, Duwi Fm., New Albany Sh., Monterey Fm., Herrin No. 6 coal, Eocene coal, and Miocene lignite from Kalimantan) were processed by density gradient centrifugation (DGC) to isolate the constituent macerals. Optimal separation, as well as the liberation of microcrystalline pyrite from the organic matter, was obtained by particle size minimization prior to DGC by treatment with liquid N2 and micronization in a fluid energy mill. The resulting small particle size limits the use of optical microscopy, thus microfluorimetry and analytical pyrolysis were also employed to assess the quality and purity of the fractions. Each of the samples exhibits one dominant DGC peak (corresponding to alginite in the Green River Fm., amorphinite in the Lower Toarcian Sh., vitrinite in the Herrin No. 6, etc.) which shifts from 1.05 g mL-1 for the Type I kerogens to between 1.18 and 1.23 g mL-1 for Type II and II-S. The characteristic densities for Type III organic matter are greater still, being 1.27 g mL-1 for the hydrogen-rich Eocene coal, 1.29 g mL-1 for the Carboniferous coal and 1.43 g mL-1 for the oxygen-rich Miocene lignite. Among Type II kerogens, the DGC profile represents a compositional continuum from undegraded alginite through (bacterial) degraded amorphinite; therefore chemical and optical properties change gradually with increasing density. The separation of useful quantities of macerals that occur in only minor amounts is difficult. Such separations require large amounts of starting material and require multiple processing steps. Complete maceral separation for some samples using present methods seems remote. Samples containing macerals with significant density differences due to heteroatom diversity (e.g., preferential sulfur or oxygen concentration in the one maceral), on the other hand, may be successfully separated (e.g., coals and

Optimized HPLC method for analysis of polar and nonpolar heterocyclic amines in cooked meat products.

PubMed

Gibis, Monika

2009-01-01

A simple, precise, and specific column high-performance liquid chromatographic (HPLC) method with UV absorption diode array and fluorescence detection has been developed by optimizing a previously described method for the simultaneous quantification of 15 polar and nonpolar heterocyclic amines (HAs) in fried meat products. The HPLC determination could be improved due to the application of a silica-based reversed-phase column with octadecyl groups (TSK-gel Super ODS) and a particle size of 2 microm. The separation of HAs in the complex meat matrix was performed with a 21 min mobile phase gradient. The method was validated for instrumental precision, repeatability, and selectivity and compared with a previously published method. After liquid adsorption of the basic sample mixture on diatomaceous earth, HAs were extracted with ethyl acetate. For cleanup, solid-phase extraction (silica propylsulfonic acid and octadecyl cartridges) and different washing steps were applied. Both nonpolar and polar HAs were determined in one fraction. The calibration curves of all HAs were linear for the applied detection system (correlation coefficient = 0.990-0.995). The recoveries, with the exception of 3-amino-1-methyl-5H-pyrido [4,3-b]indole (Trp-P-2), were between 42 and 98% from meat samples spiked in a range of 1.5 to 3.3 ng/g for fluorescence-active and 4.3 to 8 ng/g for UV-active HAs. For quantification of HAs, the standard addition method was used for adjustment of different characteristics of HAs in the extraction. In fried meat samples (chicken breast and beef patties), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5-f] quinoxaline(4,8-DiMelQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), norharmane, and harmane were found in a concentration range of 0.02 to 14.3 ng/g.

A wet/wet differential pressure sensor for measuring vertical hydraulic gradient.

PubMed

Fritz, Brad G; Mackley, Rob D

2010-01-01

Vertical hydraulic gradient is commonly measured in rivers, lakes, and streams for studies of groundwater-surface water interaction. While a number of methods with subtle differences have been applied, these methods can generally be separated into two categories; measuring surface water elevation and pressure in the subsurface separately or making direct measurements of the head difference with a manometer. Making separate head measurements allows for the use of electronic pressure sensors, providing large datasets that are particularly useful when the vertical hydraulic gradient fluctuates over time. On the other hand, using a manometer-based method provides an easier and more rapid measurement with a simpler computation to calculate the vertical hydraulic gradient. In this study, we evaluated a wet/wet differential pressure sensor for use in measuring vertical hydraulic gradient. This approach combines the advantage of high-temporal frequency measurements obtained with instrumented piezometers with the simplicity and reduced potential for human-induced error obtained with a manometer board method. Our results showed that the wet/wet differential pressure sensor provided results comparable to more traditional methods, making it an acceptable method for future use.

HPLC pigment analysis of marine phytoplankton during a red tide occurrence in Tolo Harbour, Hong Kong.

PubMed

Wong, C Kwan; Wong, C Kim

2003-09-01

A red tide was detected in the inner parts of Tolo Harbour, Hong Kong, in November 2000. Water samples were collected from a fixed station at the centre of the red tide patch for microscopic analysis of phytoplankton community composition and high performance liquid chromatography (HPLC) analysis of phytoplankton pigments. At the peak of the red tide on 24 November 2000, phytoplankton was dominated by the dinoflagellate Scrippsiella trochoidea. The red tide began to decline at the end of November and, by 1 December 2000, the phytoplankton was dominated by diatoms. Chlorophylls and carotenoids in water samples were analysed using HPLC pigment separation technique. Dinoflagellates were indicated by the signature pigment peridinin. Significant correlation (r=0.999) was found between the peridinin concentration and dinoflagellate density. A decrease in peridinin and an increase in fucoxanthin, a major carotenoid in diatoms, marked the shift in phytoplankton composition at the end of the red tide. HPLC analysis also revealed the occurrence of minor phytoplankton groups that are difficult to identify by light microscopy. Red tide monitoring and study of red tide dynamics in Hong Kong have been based on cell counting and spectrophotometric or fluorometric measurement of chlorophyll a. HPLC pigment analysis provides an effective alternative for investigating phytoplankton dynamics during red tide and other algal blooms.

Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC-ESI/MS.

PubMed

Liu, Shiming; Chen, Kaoshan; Schliemann, Willibald; Strack, Dieter

2005-01-01

A simple method involving polyamide column chromatography in combination with HPLC-PAD and HPLC-ESI/MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4 degrees C from which two main compounds were purified by semi-preparative HPLC. In comparison with the UV and ESI-MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction/separation technique was validated using an internal standard method.

[Simultaneous determination of five active constitutents in Xiaochaihu Tang by HPLC].

PubMed

Liu, Qingchun; Zhao, Junning; Yan, Liangchun; Yi, Jinhai; Song, Jun

2010-03-01

To establish a HPLC-PDA method for the determination of baicalin, wogonoside, baicalein, wogonin and glycyrrhizic acid in Xiaochaihu Tang. A Symmetry Shield RP18 (4.6 mm x 250 mm, 5.0 microm) was used with a mobile phase of acetonitrile-0.01% H3PO4 in gradient elution. The detection wavelength was 251 nm,the flow rate was 0.45 mL x min(-1) and the column temperature was maintained at 30 degrees C. The accuracy, precision, sensitivity, specificity and linearity of this method met the requirements. The contents of the five effective fractions were determined simultaneously. The method is rapid,simple and accurate and it can be suitable for the determination of baicalin, wogonoside, baicalein, wogonin and glycyrrhizic acid in Xiaochaihu Tang simultaneously.

Stability-Indicating TLC-Densitometric and HPLC Methods for the Simultaneous Determination of Piracetam and Vincamine in the Presence of Their Degradation Products.

PubMed

Ahmed, Amal B; Abdelrahman, Maha M; Abdelwahab, Nada S; Salama, Fathy M

2016-11-01

Newly established TLC-densitometric and RP-HPLC methods were developed and validated for the simultaneous determination of Piracetam (PIR) and Vincamine (VINC) in their pharmaceutical formulation and in the presence of PIR and VINC degradation products, PD and VD, respectively. The proposed TLC-densitometric method is based on the separation and quantitation of the studied components using a developing system that consists of chloroform-methanol-glacial acetic acid-triethylamine (8 + 2 + 0.1 + 0.1, v/v/v/v) on TLC silica gel 60 F254 plates, followed by densitometric scanning at 230 nm. On the other hand, the developed RP-HPLC method is based on the separation of the studied components using an isocratic elution of 0.05 M KH2PO4 (containing 0.1% triethylamine adjusted to pH 3 with orthophosphoric acid)-methanol (95 + 5, v/v) on a C8 column at a flow rate of 1 mL/min with diode-array detection at 230 nm. The developed methods were validated according to International Conference on Harmonization guidelines and demonstrated good accuracy and precision. Moreover, the developed TLC-densitometric and RP-HPLC methods are suitable as stability-indicating assay methods for the simultaneous determination of PD and VD either in bulk powder or pharmaceutical formulation. The results were statistically compared with those obtained by the reported RP-HPLC method using t- and F-tests.

Stress Degradation Studies on Varenicline Tartrate and Development of a Validated Stability-Indicating HPLC Method

PubMed Central

Pujeri, Sudhakar S.; Khader, Addagadde M. A.; Seetharamappa, Jaldappagari

2012-01-01

A simple, rapid and stability-indicating reversed-phase liquid chromatographic method was developed for the assay of varenicline tartrate (VRT) in the presence of its degradation products generated from forced decomposition studies. The HPLC separation was achieved on a C18 Inertsil column (250 mm × 4.6 mm i.d. particle size is 5 μm) employing a mobile phase consisting of ammonium acetate buffer containing trifluoroacetic acid (0.02M; pH 4) and acetonitrile in gradient program mode with a flow rate of 1.0 mL min−1. The UV detector was operated at 237 nm while column temperature was maintained at 40 °C. The developed method was validated as per ICH guidelines with respect to specificity, linearity, precision, accuracy, robustness and limit of quantification. The method was found to be simple, specific, precise and accurate. Selectivity of the proposed method was validated by subjecting the stock solution of VRT to acidic, basic, photolysis, oxidative and thermal degradation. The calibration curve was found to be linear in the concentration range of 0.1–192 μg mL−1 (R2 = 0.9994). The peaks of degradation products did not interfere with that of pure VRT. The utility of the developed method was examined by analyzing the tablets containing VRT. The results of analysis were subjected to statistical analysis. PMID:22396908

Stepwise pH-gradient elution for the preparative separation of natural anthraquinones by multiple liquid-liquid partition.

PubMed

Hynninen, P H; Räisänen, R

2001-01-01

Preparative-scale separation of substituted anthraquinones by multiple liquid-liquid partition was studied using isopropylmethyl ketone (IMK)/aqueous phosphate buffer (aq.) as the solvent system and the Hietala apparatus with 100 partition units as the partition equipment. The lower (aq.) phase was chosen as mobile, while the upper (IMK) phase remained stationary. Hence, the principle of stepwise pH-gradient elution could be utilized to separate the components in two complex mixtures of hydroxyanthraquinones and hydroxyanthraquinone carboxylic acids, isolated from the fungus Dermocybe sanguinea. In spite of the nonlinearity of the partition isotherms for these anthraquinones, implying deviations from the Nernst partition law, remarkable separations were achieved for the components in each mixture. Every anthraquinone carboxylic acid showed markedly irregular partition behavior, appearing in the effluent as two more or less resolved concentration zones. Such splitting was attributed to the formation of relatively stable sandwich-dimers, which were in a slow equilibrium with the monomers in the more nonpolar organic phase. At lower pH-values, only the polar monomers were distributed into the mobile aqueous phase and moved forward, whereas the nonpolar sandwich-dimers remained almost entirely in the stationary organic phase and lagged behind. When the pH of the mobile aqueous phase was raised high enough, the firmly linked dimers were monomerized and emerged from the apparatus as a second concentration profile. Intermolecular hydrogen bonding and pi-pi interaction between the pi-systems of two anthraquinone molecules in a parallel orientation were considered responsible for the nonlinear and markedly irregular partition behavior of the natural anthraquinones studied. The nonlinearity of the partition behavior of the hydroxyanthraquinones lacking the carboxyl group, appeared merely as excessive broadening of the experimental concentration profile, which impaired the

Separation, isolation and stereochemical assignment of imazalil enantiomers and their quantitation in an in vitro toxicity test.

PubMed

Casas, Mònica Escolà; Kretschmann, Andreas Christopher; Andernach, Lars; Opatz, Till; Bester, Kai

2016-06-24

A simple method for the separation of the enantiomers of the fungicide imazalil was developed. Racemic imazalil was separated into its enantiomers with an enantiomeric purity of 99% using HPLC-UV with an enantioselective column (permethylated cyclodextrin) operated in reversed phase mode (water with 0.2% trimethylamine and 0.08% acetic acid and methanol). The absolute configuration of the separated enantiomers was assigned and unequivocally confirmed by optical rotation as well as by vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) combined with ab-initio calculations. The same enantioselective column was also used to develop an HPLC-MS/MS method for the quantification of imazalil enantiomers. The HPLC-MS/MS method reached limits of quantification (LOQs) of 0.025mg/mL with 5μL injections. This method was used to verify imazalil concentrations and enantiomeric fractions in samples from an in vitro test on effects on human steroidogenesis (H295R steroidogenesis assay). The quantification verified the stability of the enantiomers of imazalil during the in vitro tests. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and validation of a single robust HPLC method for the characterization of a pharmaceutical starting material and impurities from three suppliers using three separate synthetic routes.

PubMed

Sheldon, E M; Downar, J B

2000-08-15

Novel approaches to the development of analytical procedures for monitoring incoming starting material in support of chemical/pharmaceutical processes are described. High technology solutions were utilized for timely process development and preparation of high quality clinical supplies. A single robust HPLC method was developed and characterized for the analysis of the key starting material from three suppliers. Each supplier used a different process for the preparation of this material and, therefore, each suppliers' material exhibited a unique impurity profile. The HPLC method utilized standard techniques acceptable for release testing in a QC/manufacturing environment. An automated experimental design protocol was used to characterize the robustness of the HPLC method. The method was evaluated for linearity, limit of quantitation, solution stability, and precision of replicate injections. An LC-MS method that emulated the release HPLC method was developed and the identities of impurities were mapped between the two methods.

Development and comparison of HPLC-MS/MS and UPLC-MS/MS methods for determining eight coccidiostats in beef.

PubMed

Zhao, Xia; Wang, Bo; Xie, Kaizhou; Liu, Jianyu; Zhang, Yangyang; Wang, Yajuan; Guo, Yawen; Zhang, Genxi; Dai, Guojun; Wang, Jinyu

2018-06-15

A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining eight coccidiostat (halofuginone, lasalocid, maduramicin, monensin, narasin, nigericin, robenidine and salinomycin) residues in beef were developed and compared. Samples were extracted with a mixture of acetic acid, acetonitrile and ethyl acetate and were then purified on a C 18 solid-phase extraction (SPE) column. The purified samples were analyzed by HPLC-MS/MS and UPLC-MS/MS, using 0.1% formic acid-water solution (A) and pure methanol (B) as the mobile phase. The samples were fractionated on a C 18 column using different gradient elution procedures, followed by qualitative analysis using a mass spectrometer operated in multiple reaction monitoring (MRM) mode with positive electrospray ionization; the external standard method was used for quantitation. At spiked levels that ranged from the limit of quantification (LOQ) to 100 μg/kg, the average recoveries were 71.96%-100.32% and 71.24%-89.24%, with relative standard deviations (RSDs) of 2.65%-12.38% and 2.98%-14.86% for UPLC-MS/MS and HPLC-MS/MS, respectively. The limits of detection (LODs) and LOQs of the eight coccidiostats were 0.14-0.32 μg/kg and 0.43-1.21 μg/kg, respectively, for UPLC-MS/MS analysis and 0.16-0.58 μg/kg and 0.53-1.92 μg/kg, respectively, for HPLC-MS/MS analysis. Both methods had good accuracy and precision, but UPLC-MS/MS had higher sensitivity than HPLC-MS/MS. Copyright © 2018 Elsevier B.V. All rights reserved.

Simultaneous determination of phenolic acids and flavonoids in rice using solid-phase extraction and RP-HPLC with photodiode array detection.

PubMed

Irakli, Maria N; Samanidou, Victoria F; Biliaderis, Costas G; Papadoyannis, Ioannis N

2012-07-01

An analytical method based on an optimized solid-phase extraction procedure and followed by high-performance liquid chromatography (HPLC) separation with diode array detection was developed and validated for the simultaneous determination of phenolic acids (gallic, protocatechuic, 4-hydroxy-benzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapic, and cinnamic acids), flavanols (catechin and epicatechin), flavonols (myricetin, quercetin, kaempferol, quercetin-3-O-glucoside, hyperoside, and rutin), flavones (luteolin and apigenin) and flavanones (naringenin and hesperidin) in rice flour (Oryza sativa L.). Chromatographic separation was carried out on a PerfectSil Target ODS-3 (250 mm × 4.6 mm, 3 μm) column at temperature 25°C using a mobile phase, consisting of 0.5% (v/v) acetic acid in water, methanol, and acetonitrile at a flow rate 1 mL min(-1) , under gradient elution conditions. Application of optimum extraction conditions, elaborated on both Lichrolut C(18) and Oasis HLB cartridges, have led to extraction of phenolic acids and flavonoids from rice flour with mean recoveries 84.3-113.0%. The developed method was validated in terms of linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 5) and inter-day precision (n = 4) revealed relative standard deviation (RSD) <13%. The optimized method was successfully applied to the analysis of phenolic acids and flavonoids in pigmented (red and black rice) and non-pigmented rice (brown rice) samples. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A validated HPLC method for the determination of pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine in rat plasma and urine.

PubMed

Abu-Qare, A W; Abou-Donia, M B

2001-12-01

A method was developed for the separation and quantification of the anti-nerve agent pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), the analgesic drugs acetaminophen and acetylsalicylic acid, and the stimulant caffeine (3,7-dihydro-1,3,7-trimethyl-1-H-purine-2,6-dione) in rat plasma and urine. The compounds were extracted using C(18) Sep-Pak(R) cartridges then analyzed by high performance liquid chromatography (HPLC) with reversed phase C18 column, and UV detection at 280 nm. The compounds were separated using gradient of 1-85% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 ml/min in a period of 14 min. The retention times ranged from 8.8 to 11.5 min. The limits of detection were ranged between 100 and 200 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 70.9+/-9.5, 73.7+/-9.8, 88.6+/-9.3, 83.9+/-7.8, and from urine 69.1+/-8.5, 74.5+/-8.7, 85.9+/-9.8, 83.2+/-9.3, for pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine, respectively. The relationship between peak areas and concentration was linear over range between 100 and 1000 ng/ml. The resulting chromatograms showed no interfering peaks from endogenous plasma or urine components. This method was applied to analyze these compounds following oral administration in rats.

Separation of cells from the rat anterior pituitary gland

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Hatfield, J. Michael

1984-01-01

Data concerned with analyzing the cellular organization of the rat anterior pituitary gland are examined. The preparation of the cell suspensions and the methods used to separate pituitary cell types are described. Particular emphasis is given to velocity sedimentation at unit gravity, density gradient centrifugation, affinity methods, fluorescence activated cell sorting, and density gradient and continuous-flow electrophoresis. The difficulties encountered when attempting to compare data from different pituitary cell separation studies are discussed, and results from various experiments are presented. The functional capabilities of the separated cell populations can be tested in various culture systems.

Retention modeling under organic modifier gradient conditions in ion-pair reversed-phase chromatography. Application to the separation of a set of underivatized amino acids.

PubMed

Pappa-Louisi, A; Agrafiotou, P; Papachristos, K

2010-07-01

The combined effect of the ion-pairing reagent concentration, C(ipr), and organic modifier content, phi, on the retention under phi-gradient conditions at different constant C(ipr) was treated in this study by using two approaches. In the first approach, the prediction of the retention time of a sample solute is based on a direct fitting procedure of a proper retention model to 3-D phi-gradient retention data obtained under the same phi-linear variation but with different slope and time duration of the initial isocratic part and in the presence of various constant C(ipr) values in the eluent. The second approach is based on a retention model describing the combined effect of C(ipr) and phi on the retention of solutes in isocratic mode and consequently analyzes isocratic data obtained in mobile phases containing different C(ipr) values. The effectiveness of the above approaches was tested in the retention prediction of a mixture of 16 underivatized amino acids using mobile phases containing acetonitrile as organic modifier and sodium dodecyl sulfate as ion-pairing reagent. From these approaches, only the first one gives satisfactory predictions and can be successfully used in optimization of ion-pair chromatographic separations under gradient conditions. The failure of the second approach to predict the retention of solutes in the gradient elution mode in the presence of different C(ipr) values was attributed to slow changes in the distribution equilibrium of ion-pairing reagents caused by phi-variation.

Continuous Flow Liquid Microjunction Surface Sampling Probe Connected On-line with HPLC/MS for Spatially Resolved Analysis of Small Molecules and Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Berkel, Gary J; Kertesz, Vilmos

RATIONALE: A continuous flow liquid microjunction surface sampling probe extracts soluble material from surfaces for direct ionization and detection by MS. Demonstrated here is the on-line coupling of such a probe with HPLC/MS enabling extraction, separation and detection of small molecules and proteins from surfaces in a spatially resolved (~0.5 mm diameter spots) manner. Methods: A continuous flow liquid microjunction surface sampling probe was connected to a 6-port, 2-position valve for extract collection and injection to an HPLC column. A QTRAP 5500 hybrid triple quadrupole linear ion trap equipped with a Turbo V ion source operated in positive ESI modemore » was used for all experiments. System operation was tested with extraction, separation and detection of propranolol and associated metabolites from drug dosed tissues and proteins from dried sheep blood spots on paper. Results: Confirmed in the tissue were the parent drug and two different hydroxypropranolol glucuronides. The mass spectrometric response for these compounds from different locations in the liver showed an increase with increasing extraction time (5, 20 and 40 s extractions). For on-line separation and detection/identification of extracted proteins from dried sheep blood spots, two major protein peaks dominated the chromatogram and could be correlated with the expected masses for the hemoglobin and chains. Conclusions: Spatially resolved sampling, separation, and detection of small molecules and proteins from surfaces can be accomplished using a continuous flow liquid microjunction surface sampling probe coupled on-line with HPLC/MS detection.« less

Identification and quantification of 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones in Chinese eaglewood by HPLC with diode array detection and MS.

PubMed

Li, Jun; Chen, Dong; Jiang, Yong; Zhang, Qian; Zhang, Liang; Tu, Peng-Fei

2013-12-01

A sensitive and reliable HPLC coupled with diode array detection and MS method was developed and validated for the first time to simultaneously identify and quantify eight characteristic 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones (THPECs) in Chinese eaglewood. Chromatographic separation was performed on a Zorbax SB C18 column with a gradient of acetonitrile/0.1% formic acid/water as the mobile phase. The MS fragmentation behavior of THPECs was characterized as the successive neutral loss of two molecules of H2 O ([M+H-18-18](+) ) and then two molecules of CO ([M+H-18-18-28-28](+) ), which could be used to differentiate Chinese eaglewood from counterfeits. Validation of the developed analytical method showed good linearity, satisfactory precision, and good recovery. The established method was successfully applied to the simultaneous determination of eight THPECs in ten batches of Chinese eaglewood, which could be used as a tool for the quality control of Chinese eaglewood. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[HPLC Specific Fingerprint of Alcohol Extract of Andrographis paniculata].

PubMed

Liu, Fei-fei; Fan, Chun-lin; Huang, Xiao-jun; Wang, Gui-yang; Wang, Ying; Ye, Wen-cai

2015-07-01

To establish the specific fingerprint of alcohol extract of Andrographis paniculata by HPLC. The analysis was performed on Cosmosil 5C18 -MS-II (250 mm x 4. 6 mm, 5 µm) column, with gradient phase consisting of acetonitrile and water at the flow rate of 1. 0 mL/min. The UV detection wavelength was set at 225 nm, and column temperature was 30 °C. The specific fingerprint chromatogram was established and seven common peaks were identified by comparison with the reference standards and LC-MS. The relative retention times were 1. 00 (No. 1, andrographolide), 1. 04 (No. 2, deoxyandrographoside), 1. 07 (No. 3, isoandrographolide), 1. 10 (No. 4, 14-deoxy-11, 12-didehydroandrographoside), 1. 50 (No. 5, neoandrographolide), 1. 75 ( No. 6, deoxyandrographolide) and 1. 79 (No. 7, dehydroandrographolide). The method is simple and reproducible with high precision, which can provide the basis for the quality control and evaluation of alcohol extract of Andrographis paniculata and its preparations.

Validation of a method for the quantitation of ghrelin and unacylated ghrelin by HPLC.

PubMed

Staes, Edith; Rozet, Eric; Ucakar, Bernard; Hubert, Philippe; Préat, Véronique

2010-02-05

An HPLC/UV method was first optimized for the separation and quantitation of human acylated and unacylated (or des-acyl) ghrelin from aqueous solutions. This method was validated by an original approach using accuracy profiles based on tolerance intervals for the total error measurement. The concentration range that achieved adequate accuracy extended from 1.85 to 59.30microM and 1.93 to 61.60microM for acylated and unacylated ghrelin, respectively. Then, optimal temperature, pH and buffer for sample storage were determined. Unacylated ghrelin was found to be stable in all conditions tested. At 37 degrees C acylated ghrelin was stable at pH 4 but unstable at pH 7.4, the main degradation product was unacylated ghrelin. Finally, this validated HPLC/UV method was used to evaluate the binding of acylated and unacylated ghrelin to liposomes.

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations.

PubMed

Tölgyesi, Ádám; Barta, Enikő; Simon, Andrea; McDonald, Thomas J; Sharma, Virender K

2017-10-25

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed for their applications in livestock of the European Union (EU). This paper presents analyses of twelve selected steroids and six nitroimidazole antibiotics at low levels (1.56μg/L-4.95μg/L and 0.17μg/kg-2.14μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up procedures, high performance liquid chromatography (HPLC) separation, and tandem mass spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples were cleaned by two independent supported liquid extraction and solid phase extraction procedures. Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost effective clean-up. The confirmatory methods were improved by extending the number of matrices and compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new methods were validated according to the recommendation of the European Union Reference Laboratories and the performance characteristics evaluated met fully the criteria. The methods were applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the applicability of developed protocols of the methods to real samples. The confirmatory methods were applied to the national monitoring program and natural contamination of prednisolone could be detected in urine at low concentration in few samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Separation of charge-regulated polyelectrolytes by pH-assisted diffusiophoresis.

PubMed

Hsu, Jyh-Ping; Hsu, Yen-Rei; Shang-Hung, Hsieh; Tseng, Shiojenn

2017-03-29

The potential of separating colloidal particles through simultaneous application of a salt gradient and a pH gradient, or pH-assisted diffusiophoresis, is evaluated by considering the case of spherical polyelectrolytes (PEs) having different equilibrium dissociation constants in an aqueous solution with KCl as the background salt. The simulation results gathered reveal that the dependence of the particle velocity on pH is more sensitive than that in pH-assisted electrophoresis, where an electric field and a pH gradient are applied simultaneously. This implies that the separation efficiency of pH-assisted diffusiophoresis can be better than that of pH-assisted electrophoresis. In particular, two types of PE having different equilibrium dissociation constants can be separated effectively by applying the former by enhancing/reducing their diffusiophoretic velocities.

[Fast optimization of stepwise gradient conditions for ternary mobile phase in reversed-phase high performance liquid chromatography].

PubMed

Shan, Yi-chu; Zhang, Yu-kui; Zhao, Rui-huan

2002-07-01

In high performance liquid chromatography, it is necessary to apply multi-composition gradient elution for the separation of complex samples such as environmental and biological samples. Multivariate stepwise gradient elution is one of the most efficient elution modes, because it combines the high selectivity of multi-composition mobile phase and shorter analysis time of gradient elution. In practical separations, the separation selectivity of samples can be effectively adjusted by using ternary mobile phase. For the optimization of these parameters, the retention equation of samples must be obtained at first. Traditionally, several isocratic experiments are used to get the retention equation of solute. However, it is time consuming especially for the separation of complex samples with a wide range of polarity. A new method for the fast optimization of ternary stepwise gradient elution was proposed based on the migration rule of solute in column. First, the coefficients of retention equation of solute are obtained by running several linear gradient experiments, then the optimal separation conditions are searched according to the hierarchical chromatography response function which acts as the optimization criterion. For each kind of organic modifier, two initial linear gradient experiments are used to obtain the primary coefficients of retention equation of each solute. For ternary mobile phase, only four linear gradient runs are needed to get the coefficients of retention equation. Then the retention times of solutes under arbitrary mobile phase composition can be predicted. The initial optimal mobile phase composition is obtained by resolution mapping for all of the solutes. A hierarchical chromatography response function is used to evaluate the separation efficiencies and search the optimal elution conditions. In subsequent optimization, the migrating distance of solute in the column is considered to decide the mobile phase composition and sustaining time of the

Determination of fenoterol in human plasma by HPLC with fluorescence detection after derivatization.

PubMed

Meineke, Ingolf; Steinmetz, Hannelore; Kramer, Skaidrit; Gleiter, Christoph H

2002-06-20

A new method for the determination of fenoterol is described, which uses HPLC separation with fluorescence detection. Dobutamine is employed as an internal standard. The separation was achieved on a short reversed phase column with a mobile phase consisting of water, acetonitrile and methanol. Prior to chromatography both analytes are derivatized with 9-chloroformyl-carbazole. Isolation of the analytes from plasma is carried out by liquid-liquid extraction into 2-butanol after protein precipitation with acetonitrile. The method is capable of estimating fenoterol concentrations in the sub-nanogram per ml range with sufficient accuracy and precision. The determination of fenoterol can now be carried out in the average laboratory without radiolabelled material.

Microfluidic immunomagnetic cell separation from whole blood.

PubMed

Bhuvanendran Nair Gourikutty, Sajay; Chang, Chia-Pin; Puiu, Poenar Daniel

2016-02-01

Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of a 45kpsi ultrahigh pressure liquid chromatography instrument for gradient separations of peptides using long microcapillary columns and sub-2μm particles.

PubMed

Grinias, Kaitlin M; Godinho, Justin M; Franklin, Edward G; Stobaugh, Jordan T; Jorgenson, James W

2016-10-21

Commercial chromatographic instrumentation for bottom-up proteomics is often inadequate to resolve the number of peptides in many samples. This has inspired a number of complex approaches to increase peak capacity, including various multidimensional approaches, and reliance on advancements in mass spectrometry. One-dimensional reversed phase separations are limited by the pressure capabilities of commercial instruments and prevent the realization of greater separation power in terms of speed and resolution inherent to smaller sorbents and ultrahigh pressure liquid chromatography. Many applications with complex samples could benefit from the increased separation performance of long capillary columns packed with sub-2μm sorbents. Here, we introduce a system that operates at a constant pressure and is capable of separations at pressures up to 45kpsi. The system consists of a commercially available capillary liquid chromatography instrument, for sample management and gradient creation, and is modified with a storage loop and isolated pneumatic amplifier pump for elevated separation pressure. The system's performance is assessed with a complex peptide mixture and a range of microcapillary columns packed with sub-2μm C18 particles. Copyright © 2016 Elsevier B.V. All rights reserved.

Systematic Interpolation Method Predicts Antibody Monomer-Dimer Separation by Gradient Elution Chromatography at High Protein Loads.

PubMed

Creasy, Arch; Reck, Jason; Pabst, Timothy; Hunter, Alan; Barker, Gregory; Carta, Giorgio

2018-05-29

A previously developed empirical interpolation (EI) method is extended to predict highly overloaded multicomponent elution behavior on a cation exchange (CEX) column based on batch isotherm data. Instead of a fully mechanistic model, the EI method employs an empirically modified multicomponent Langmuir equation to correlate two-component adsorption isotherm data at different salt concentrations. Piecewise cubic interpolating polynomials are then used to predict competitive binding at intermediate salt concentrations. The approach is tested for the separation of monoclonal antibody monomer and dimer mixtures by gradient elution on the cation exchange resin Nuvia HR-S. Adsorption isotherms are obtained over a range of salt concentrations with varying monomer and dimer concentrations. Coupled with a lumped kinetic model, the interpolated isotherms predict the column behavior for highly overloaded conditions. Predictions based on the EI method showed good agreement with experimental elution curves for protein loads up to 40 mg/mL column or about 50% of the column binding capacity. The approach can be extended to other chromatographic modalities and to more than two components. This article is protected by copyright. All rights reserved.

Simultaneous separation and quantitation of amino acids and polyamines of forest tree tissues and cell cultures within a single high-performance liquid chromatography run using dansyl derivatization

Treesearch

Rakesh Minocha; Stephanie Long

2004-01-01

The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures. The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl...

The role of cell size in density gradient electrophoretic separation of mouse leukemia cells according to position in the cell cycle

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

Cultured mouse leukemia cells line L5178Y were subjected to upward electrophoresis in a density gradient and the slower migrating cell populations were enriched in G2 cells. It is indicated that this cell line does not change electrophoretic mobility through the cell cycle. The possibility that increased sedimentation downward on the part of the larger G2 cells caused this separation was explored. Two different cell populations were investigated. The log phase population was found to migrate upward faster than the G2 population, and a similar difference between their velocities and calculated on the basis of a 1 um diameter difference between the two cell populations. The G2 and G1 enriched populations were isolated by Ficoll density gradient sedimentation. The bottom fraction was enriched in G2 cells and the top fraction was enriched with G1 cells, especially when compared with starting materials. The electrophoretic mobilities of these two cell populations did not differ significantly from one another. Cell diameter dependent migration curves were calculated and were found to be different. Families of migration curves that differ when cell size is considered as a parameter are predicted.

Magnetophoresis of iron oxide nanoparticles at low field gradient: the role of shape anisotropy.

PubMed

Lim, Jitkang; Yeap, Swee Pin; Leow, Chee Hoe; Toh, Pey Yi; Low, Siew Chun

2014-05-01

Magnetophoresis of iron oxide magnetic nanoparticle (IOMNP) under low magnetic field gradient (<100 T/m) is significantly enhanced by particle shape anisotropy. This unique feature of magnetophoresis is influenced by the particle concentration and applied magnetic field gradient. By comparing the nanosphere and nanorod magnetophoresis at different concentration, we revealed the ability for these two species of particles to achieve the same separation rate by adjusting the field gradient. Under cooperative magnetophoresis, the nanorods would first go through self- and magnetic field induced aggregation followed by the alignment of the particle clusters formed with magnetic field. Time scale associated to these two processes is investigated to understand the kinetic behavior of nanorod separation under low field gradient. Surface functionalization of nanoparticles can be employed as an effective strategy to vary the temporal evolution of these two aggregation processes which subsequently influence the magnetophoretic separation time and rate. Copyright © 2014 Elsevier Inc. All rights reserved.

Gradient stationary phase optimized selectivity liquid chromatography with conventional columns.

PubMed

Chen, Kai; Lynen, Frédéric; Szucs, Roman; Hanna-Brown, Melissa; Sandra, Pat

2013-05-21

Stationary phase optimized selectivity liquid chromatography (SOSLC) is a promising technique to optimize the selectivity of a given separation. By combination of different stationary phases, SOSLC offers excellent possibilities for method development under both isocratic and gradient conditions. The so far available commercial SOSLC protocol utilizes dedicated column cartridges and corresponding cartridge holders to build up the combined column of different stationary phases. The present work is aimed at developing and extending the gradient SOSLC approach towards coupling conventional columns. Generic tubing was used to connect short commercially available LC columns. Fast and base-line separation of a mixture of 12 compounds containing phenones, benzoic acids and hydroxybenzoates under both isocratic and linear gradient conditions was selected to demonstrate the potential of SOSLC. The influence of the connecting tubing on the deviation of predictions is also discussed.

COMPUTER-ASSISTED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT WITH APPLICATIONS TO THE ISOLATION AND ANALYSIS OF PHYTOPLANKTON PIGMENTS. (R826944)

EPA Science Inventory

We used chromatography modeling software to assist in HPLC method development, with the goal
of enhancing separations through the exclusive use of gradient time and column temperature. We
surveyed nine stationary phases for their utility in pigment purification and natur...

Diversity oriented high-throughput screening of 1,3,4-oxadiazole modified chlorophenylureas and halogenobenzamides by HPLC with peptidomimetic calixarene-bonded stationary phases.

PubMed

Bazylak, Grzegorz; Malak, Anna; Ali, Imran; Borowiak, Teresa; Dutkiewicz, Grzegorz

2008-06-01

Retention profiles in series of the neutral and highly hydrophobic 1,3,4-oxadiazoles containing chlorophenylurea and halogenobenzamide moiety and indicating analgesic activity were determined in the isocratic standard- and narrow-bore HPLC systems employing, respectively, various octadecylsilica and different calixarene bonded stationary phases. When acetonitrile - 2.65 mM phosphoric acid (55 : 45, %, v/v), pH* 3.25, mobile phase was applied retention of these compounds increased with decline of their overall hydrophobicity according to the general preference of more polar compounds by calixarene cavity in time of its non-specific host-guest supramolecular interactions with halogenated substances. The size of calixarene nanocavity and its upper-rim substitution did not change the observed retention order, resolution and selectivity of separation for oxadiazoles. Compared to the retention on the non-end-capped and the highly-end-capped octadecylsilica HPLC column a most improved separation of some regioisomers of halogenated 1,3,4-oxadiazoles were observed on both used calixarene-type HPLC supports. In addition, preliminary data on the self-assembled supramolecular crystal structure of exemplary 1,3,4-oxadiazolchlorophenylurea with cis-elongated conformation was reported and formation of the monovalent inclusion host-guest complexes between 1,3,4-oxadiazoles and each calixarene-type stationary phase was studied with molecular modelling MM+ and AM1 methods. The structural, isomeric and energetic factors leading to the hydrogen bond stabilized inclusion complexes between these species were considered and used for explanation of observed retention sequence and selectivity of 1,3,4-oxadiazoles separation in applied calixarene-based HPLC systems. All these data would be useful in future development of optimized procedures enabling encapsulation of 1,3,4-oxadiazolurea-type drugs with calixarenes.

Quality evaluation and pattern recognition analyses of marker compounds from five medicinal drugs of Rutaceae family by HPLC/PDA.

PubMed

Zhao, Bing Tian; Kim, Eun Jung; Son, Kun Ho; Son, Jong Keun; Min, Byung Sun; Woo, Mi Hee

2015-08-01

To establish a standard of quality control and to identify different origins for the Rutaceae family [Citri Unshiu Peel (CU), Citri Unshiu Immature Peel (CI), Ponciri Immature Fructus (PI), Aurantii Immature Fructus (AI), and Aurantii Fructus (AU)], 13 standards including rutin (1), narirutin (2), naringin (3), hesperidin (4), neohesperidin (5), neoponcirin (6), poncirin (7), naringenin (8), isosinensetin (9), sinensetin (10), nobiletin (11), heptamethoxyflavone (12), and tangeretin (13) were determined by high performance liquid chromatography (HPLC)/photo-diode array (PDA) analysis. A YMC ODS C18 (250 × 4.6 mm, 5 µm) column was used and the ratio of mobile phases of water (A) and acetonitrile (B) delivered to the column for gradient elution was applied. This method was fully validated with respect to linearity, accuracy, precision, stability, and robustness. The HPLC/PDA method was applied successfully to quantify 13 major compounds in the extracts of CU, CI, PI, AI, and AU. The pattern recognition analysis combined with LC chromatographic data was performed by repeated analysis of 27 reference samples in the above five Rutaceae oriental medicinal drugs. The established HPLC method was rapid and reliable for quantitative analysis and quality control of multiple components in five Rutaceae species with different origins.

[Simultaneous determination of seven constituents in Euodiae Fructus and two related species by HPLC].

PubMed

Yin, Yuan-Yuan; Yan, Li-Hua; Zhang, Qi-Wei; Zhang, Yong-Xin; Lin, Li-Mei; Zhang, Shan-Shan; Wang, Zhi-Min

2014-07-01

This study is to develop a HPLC method for quality evaluation of Euodiae Fructus and related species by simultaneous determination limonin, indole alkaloids (14-fomyldihydroxyrutaecarpine, evodiamine, rutaecarpine), and quinolone alkaloids [1-methyl-2-undecyl-4 (1H)-quinolone, evocarpine, dihydroevocarpine] in the fruits of five Evodia species. Samples were analyzed on a YMC C18 column (4.6 mm x 250 mm, 5 microm) eluted with mobile phases of acetonitrile (A), tetrahydrofuran (B), and a buffer solution of 5 mmol x L(-1) ammonium acetate (pH 3.8) (C) in a linear gradient mode. The column temperature was 30 degrees C and the flow rate was 1.0 mL x min(-1). The PDA detector wavelengths were set at 220 and 250 nm. The seven compounds were well separated and showed good linearity (r = 0.999 9) within the concentration ranges tested. The mean recoveries were between 96.7%-102.4% (RSD 1.4%-3.1%). Through the validation, the method was proved to be accurate and repeatable. All the seven constituents were detected in the fruits of five species, but the contents of them varied widely in different samples. The total contents of seven constituents in 16 batches of Euodiae Fructus were 9.46-69.9 mg x g(-1), and the mean content was 28.2 mg x g(-1). The total content of seven constituents in E. compacta and E. fargesii was 25.8, 7.69 mg x g(-1), respectively.

Quantification of active principles and pigments in leaf extracts of Isatis tinctoria by HPLC/UV/MS.

PubMed

Mohn, Tobias; Potterat, Olivier; Hamburger, Matthias

2007-02-01

An HPLC method has been developed and validated for the quantification of the pharmacologically active principles tryptanthrin (1), 1,3-dihydro-3-[(4-hydroxy-3,5-dimethoxyphenyl)methylene]-2 H-indol-2-one (indolinone) (3), indirubin (4), alpha-linolenic acid (2), and indigo (5), an isomer of indirubin, in extracts from the traditional anti-inflammatory plant Isatis tinctoria (woad). The chromatographic separation was performed on a C-18 column with a linear gradient of acetonitrile in water containing 0.1% formic acid. The method combines UV and electrospray MS detection in the positive ion mode for the detection of the alkaloids, with a switch to the negative mode for the analysis of alpha-linolenic acid. The method was applied to the analysis of woad extracts obtained by supercritical fluid (SFE) CO2 extraction, and by pressurized liquid extraction (PLE) with dichloromethane and methanol, respectively. While the highest concentration of alpha-linolenic acid was found in the SFE extract (7.43%), the concentrations of tryptanthrin , indolinone, indirubin and indigo were the highest in the dichloromethane extract (0.30, 0.035, 2.48 and 0.84%, respectively). Compound 3 was not detected in the methanolic extract and only traces of compounds 1, 4 and 5 and low amount of alpha-linolenic acid (0.39%) were present in this extract.

Turbulent flow separation in three-dimensional asymmetric diffusers

NASA Astrophysics Data System (ADS)

Jeyapaul, Elbert

2011-12-01

Turbulent three-dimensional flow separation is more complicated than 2-D. The physics of the flow is not well understood. Turbulent flow separation is nearly independent of the Reynolds number, and separation in 3-D occurs at singular points and along convergence lines emanating from these points. Most of the engineering turbulence research is driven by the need to gain knowledge of the flow field that can be used to improve modeling predictions. This work is motivated by the need for a detailed study of 3-D separation in asymmetric diffusers, to understand the separation phenomena using eddy-resolving simulation methods, assess the predictability of existing RANS turbulence models and propose modeling improvements. The Cherry diffuser has been used as a benchmark. All existing linear eddy-viscosity RANS models k--o SST,k--epsilon and v2- f fail in predicting such flows, predicting separation on the wrong side. The geometry has a doubly-sloped wall, with the other two walls orthogonal to each other and aligned with the diffuser inlet giving the diffuser an asymmetry. The top and side flare angles are different and this gives rise to different pressure gradient in each transverse direction. Eddyresolving simulations using the Scale adaptive simulation (SAS) and Large Eddy Simulation (LES) method have been used to predict separation in benchmark diffuser and validated. A series of diffusers with the same configuration have been generated, each having the same streamwise pressure gradient and parametrized only by the inlet aspect ratio. The RANS models were put to test and the flow physics explored using SAS-generated flow field. The RANS model indicate a transition in separation surface from top sloped wall to the side sloped wall at an inlet aspect ratio much lower than observed in LES results. This over-sensitivity of RANS models to transverse pressure gradients is due to lack of anisotropy in the linear Reynolds stress formulation. The complexity of the flow

Enantioselective determination of metoprolol and its metabolites in human urine high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and tandem mass spectrometry (MS/MS).

PubMed

Baranowska, Irena; Adolf, Weronika; Magiera, Sylwia

2015-11-01

A sensitive, stereoselective assay using solid phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) was developed and validated for the analysis of enantiomers of metoprolol and its metabolites (α-hydroxymetoprolol, O-desmethylmetoprolol). Chiral separation was achieved using a CHIRALCEL OD-RH column, packed with cellulose tris-(3,5-dimethylphenyl-carbamate) stationary phase, employing a mobile phase composed by a mixture of 0.2% diethylamine in water and acetonitrile in gradient elution mode. Linear calibration curves were obtained over the range of 0.025-2.0μg/mL (R(2)>0.994) in urine for both enantiomers of metoprolol and its metabolites with quantitation limit of 0.025μg/mL. Intra and inter-day precision and accuracy were below 15% for both metoprolol and metabolites enantiomers. The recovery of enantiomer of metoprolol and its metabolite was greater than 68.0%, utilizing a SPE procedure. The method was tested with urine quality control samples and human urine fractions after administration of 50mg rac-metoprolol. Copyright © 2015 Elsevier B.V. All rights reserved.

[Determination of sennosides and degraded products in the process of sennoside metabolism by HPLC].

PubMed

Sun, Yan; Li, Xuetuo; Yu, Xingju

2004-01-01

A method for the separation and determination of sennosides A and B and the main composition (sennidins A and B) in degraded products of sennosides by linear gradient high performance liquid chromatography has been developed. Separation conditions were as follows: column, a Spherisorb C18 column (250 mm x 4.6 mm i.d., 10 microm); column temperature, 40 degrees C; detection wavelength, 360 nm; mobile phase A, 1.25% acetic acid aqueous solution; mobile phase B, methanol; linear gradient, 100% A --> (20 min) 100% B. The method is effective, quick, accurate and reproducible. The satisfactory results show that this new method has certain practical values as an approach of real-time analysis in the process of sennoside metabolism.

Modeling of salt and pH gradient elution in ion-exchange chromatography.

PubMed

Schmidt, Michael; Hafner, Mathias; Frech, Christian

2014-01-01

The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion-exchange columns is a well-established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO3 (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well-characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.