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Sample records for gradient hplc separation

  1. An Inexpensive Digital Gradient Controller for HPLC.

    ERIC Educational Resources Information Center

    Brady, James E.; Carr, Peter W.

    1983-01-01

    Use of gradient elution techniques in high performance liquid chromatography (HPLC) is often essential for direct separation of complex mixtures. Since most commercial controllers have features that are of marginal value for instructional purposes, a low-cost controller capable of illustrating essential features of gradient elution was developed.…

  2. Performance of HPLC/MS microchips in isocratic and gradient elution modes.

    PubMed

    Ehlert, Steffen; Trojer, Lukas; Vollmer, Martin; van de Goor, Tom; Tallarek, Ulrich

    2010-03-01

    We analyzed the chromatographic performance of particle-packed, all-polyimide high-performance liquid chromatography/mass spectrometry (HPLC/MS) microchips in terms of their hydraulic permeabilities and separation efficiency under isocratic and gradient elution conditions. The separation channels of the chips (with ca 50 microm x 75 microm trapezoidal cross-section and a length of 43 mm) were slurry packed with either 3.5 or 5 microm spherical porous C18-silica particles. A custom-built holder enveloped the chip during packing to prevent channel deformation and delamination from high pressures. It is shown that the packing conditions significantly impact the packing density of the HPLC/MS chips, which determines their performance in both, isocratic and gradient elution modes. Even with steep solvent gradients, peak shape and chromatographic resolution for the densely packed HPLC/MS chips are much improved. Our data show that the analytical power of the HPLC/MS chip is limited by the quality of the chromatographic separation. PMID:20209581

  3. Gradient Scouting in Reversed-Phase HPLC Revisited

    ERIC Educational Resources Information Center

    Alcazar, A.; Jurado, J. M.; Gonzalez, A. G.

    2011-01-01

    Gradient scouting is the best way to decide the most suitable elution mode in reversed-phase high-performance liquid chromatography (RP-HPLC). A simple rule for this decision involves the evaluation of the ratio [delta]t/t[subscript G] (where [delta]t is the difference in the retention time between the last and the first peak and t[subscript G] is…

  4. Optimizing Chromatographic Separation: An Experiment Using an HPLC Simulator

    ERIC Educational Resources Information Center

    Shalliker, R. A.; Kayillo, S.; Dennis, G. R.

    2008-01-01

    Optimization of a chromatographic separation within the time constraints of a laboratory session is practically impossible. However, by employing a HPLC simulator, experiments can be designed that allow students to develop an appreciation of the complexities involved in optimization procedures. In the present exercise, a HPLC simulator from "JCE…

  5. Separation of kafirins on surface porous RP-HPLC columns

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Surface porous HPLC columns were investigated for the separation of kafarins, storage proteins of grain sorghum. Kafirins were successfully separated using C3, C8 and C18 surface porous stationary phases in less than 17 min. Separations using a monolithic C18 stationary phase were also developed ...

  6. Gradient HPLC-DAD determination of paracetamol, phenylephrine hydrochloride, cetirizine in tablet formulation.

    PubMed

    Dewani, A P; Shelke, P G; Bakal, R L; Jaybhaye, S S; Chandewar, A V; Patra, S

    2014-05-01

    Present work describes the development and validation of a simple and reliable high-performance liquid chromatography-diode array detection (HPLC-DAD) procedure for the analysis of phenylephrine hydrochloride (PHE), paracetamol (PAR) and cetirizine dihydrochloride (CET), in pharmaceutical mixture. The method was applied successfully on tablet dosage form. Effective chromatographic separation of PHE, PAR and CET was achieved using a Kinetex-C18 (4.6, 150 mm, 5 mm) column with gradient elution of the mobile phase composed of 10 mM phosphate buffer (pH 3.3) and acetonitrile. The elution was a 3 step gradient elution program step-1 started initially with 2% (by volume) acetonitrile and 98% phosphate buffer (pH 3.3) for first 2 min. In step-2 acetonitrile concentration changed linearly to 20% upto 12 min the analysis was concluded by step-3 changing acetonitrile to 2% upto 20 min. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity and robustness. Calibration curves were linear in the ranges 5-15, 250-750 and 2.5-7.5 μg/ml for PHE, PAR and CET, with correlation coefficients >0.9996. The validated HPLC method was applied to a pharmaceutical mixture of a marketed preparation tablet in which the analytes were successfully quantified with good recovery values with no interfering peaks from the excipents. PMID:24132702

  7. Gradient HPLC-DAD determination of two pharmaceutical mixtures containing the antihistaminic drug ebastine.

    PubMed

    Haggag, Rim S; Belal, Tarek S

    2012-01-01

    This work describes the development, validation and application of a simple and reliable high-performance liquid chromatography-diode array detection (HPLC-DAD) procedure for the analysis of two pharmaceutical mixtures. The first mixture contains the antihistaminic drug ebastine (EBS) and the famous sympathomimetic drug pseudoephedrine hydrochloride (PSD), and the second mixture is composed of EBS and another sympathomimetic agent, phenylephrine hydrochloride (PHR). Effective chromatographic separation of EBS, PSD and PHR was achieved using a Zorbax SB-C8 (4.6 × 250 mm, 5 μm) column with gradient elution of the mobile phase composed of 0.05M phosphoric acid and acetonitrile. The gradient elution started with 20% (by volume) acetonitrile, ramped up linearly to 90% in 5 min, then kept constant until the end of the run. The mobile phase was pumped at a flow rate of 1 mL/min. The multiple wavelength detector was set at 254 (for EBS and PSD) and 274 nm (for PHR) and quantification of the analytes was based on measuring their peak areas. The retention times for PHR, PSD and EBS were approximately 2.5, 2.9 and 7.1 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness and detection and quantification limits. Calibration curves were linear in the ranges 5-100, 100-1,000 and 10-200 µg/mL for EBS, PSD and PHR, respectively, with correlation coefficients > 0.9996. The validated HPLC method was applied to the analysis of the two pharmaceutical mixtures in laboratory-made tablets in which the analytes were successfully quantified with good recovery values and no interfering peaks were encountered from the inactive ingredients. Finally, the proposed method made use of DAD as a tool for peak identity and purity confirmation. PMID:22677488

  8. A laboratory superconducting high gradient magnetic separator

    SciTech Connect

    Yan, L.G.; Yu, Y.J.; Wang, Z.K.; Kao, Z.Y.; Ye, Z.X.; Xue, C.L.; Ye, P.; Cheng, Y.L.; Li, X.M.; Kong, Q.M.

    1989-03-01

    In order to know the effectiveness of high gradient magnetic separation for Kaolin clay purification and coal desulfurization in China and to develop suitable technology, a superconducting HGMS facility has been constructed and put into operation at the Institute of Electrical Engineering of Chinese Academy of Sciences. The working separation chamber is 80mm in diameter and 400mm in length. the magnet is wound with 0.75 and 0.5 mm in diameter NbTi superconducting composite. The winding is compact and wax-filled. The test proves that the magnet can operate at 5T. Special attention has been paid in the design and construction of the magnet cryostat in order for it to work as long as possible. In the wet beneficiation mode, there are two separation systems available, one is the upward pumping feeding system and another is with the downward gravity feeding. The rate of flow and the linear velocity are 0-0.5L/s and 0-100 cm/s respectively. The preliminary sample test results for Kaolin clay purification and coal desulfurization show the good feasibility of magnetic separation.

  9. Separation and quantitative analysis of alkyl sulfate ethoxymers by HPLC.

    PubMed

    Morvan, Julien; Hubert-Roux, Marie; Agasse, Valérie; Cardinael, Pascal; Barbot, Florence; Decock, Gautier; Bouillon, Jean-Philippe

    2008-01-01

    Separation of alkyl sulfate ethoxymers is investigated on various high-performance liquid chromatography (HPLC) stationary phases: Acclaim C18 Surfactant, Surfactant C8, and Hypercarb. For a fixed alkyl chain length, ethoxymers are eluted in the order of increasing number of ethoxylated units on Acclaim C18 Surfactant, whereas a reversed elution order is observed on Surfactant C8 and Hypercarb. Moreover, on an Acclaim C18 Surfactant column, non-ethoxylated compounds are eluted in their ethoxymers distribution and the use of sodium acetate additive in mobile phase leads to a co-elution of ethoxymers. HPLC stationary phases dedicated to surfactants analysis are evaluated by means of the Tanaka test. Surfactant C8 presents a great silanol activity whereas Acclaim C18 Surfactant shows a high steric selectivity. For alkyl sulfates, linearity of the calibration curve and limits of detection and quantitation are evaluated. The amount of sodium laureth sulfate raw material found in commercial body product is in agreement with the specification of the manufacturer. PMID:19007494

  10. The effects of eluent's optothermal parameters on TLS detection in gradient HPLC

    NASA Astrophysics Data System (ADS)

    Kožar Logar, J.; Franko, M.

    2003-01-01

    The effects of changing solvent composition on the limits of detection (LOD) of thermal lens spectroscopy (TLS) detection in gradient elution HPLC have been studied from the perspective of solvent's thermooptical properties. The refractive index and the temperature dependence of refractive index were measured and are reported for the first time for the methanol-acetone and methanol-ammonium acetate solutions used in this work, and some other methanol based chromatographic eluents. The TLS signal noise during a gradient HPLC protocol is increasing and its value reaches the maximum with a considerable time delay (up to 3 min) compared to the end point of the gradient protocol. The length of the chromatographic column was found to be an important parameter in reduction of signal noise resulting from incomplete solvent mixing. For some carotenoids the LOD values on the order of 50-100 pg/mL can be obtained on a 25 cm column with solvents that yield an increase in TLS enhancement factor up to 1.6 times during the gradient HPLC analysis.

  11. Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts.

    PubMed

    Urakova, Irina N; Pozharitskaya, Olga N; Shikov, Alexander N; Kosman, Vera M; Makarov, Valery G

    2008-02-01

    Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means. PMID:18183554

  12. Chiral HPLC separation and CD spectra of the enantiomers of the alkaloid tacamonine and related compounds.

    PubMed

    Caccamese, S; Principato, G; Jokela, R; Tolvanen, A; Din Belle, D

    2001-01-01

    The HPLC enantiomeric separation of racemic indole alkaloids tacamonine, 17 alpha-hydroxytacamonine, deethyleburnamonine, and vindeburnol was accomplished using Chiralpak AD and Chiralcel OD as chiral stationary phases. Small structural differences affect the enantioselectivity ability of these phases. Single enantiomers of tacamonine and vindeburnol were isolated by semipreparative HPLC and their CD spectra and optical rotations were measured. PMID:11746802

  13. Thermoacoustic mixture separation with an axial temperature gradient

    SciTech Connect

    Geller, Drew W; Swift, Gregory A

    2008-01-01

    The theory of thermoacoustic mixture separation is extended to include the effect of a nonzero axial temperature gradient. The analysis yields a new term in the second-order mole flux that is proportional to the temperature gradient and to the square of the volumetric velocity and is independent of the phasing of the wave. Because of this new term, thermoacoustic separation stops at a critical temperature gradient and changes direction above that gradient. For a traveling wave, this gradient is somewhat higher than that predicted by a simple four-step model. An experiment tests the theory for temperature gradients from 0 to 416 K/m in 50-50 He-Ar mixtures.

  14. Phase separation in thin films: Effect of temperature gradients

    NASA Astrophysics Data System (ADS)

    Jaiswal, Prabhat K.; Puri, Sanjay; Binder, Kurt

    2013-09-01

    We study the phase-separation kinetics of a binary (AB) mixture confined in a thin film of thickness D with a temperature gradient. Starting from a Kawasaki-exchange kinetic Ising model, we use a master-equation approach to systematically derive an extension of the Cahn-Hilliard model for this system. We study the effect of temperature gradients perpendicular to the film with “neutral” (no preference for either A or B) surfaces. We highlight the rich phenomenology and pattern dynamics which arises from the interplay of phase separation and the temperature gradient.

  15. Extraction, Separation, and Identification of Phenolic Compounds in Virgin Olive Oil by HPLC-DAD and HPLC-MS

    PubMed Central

    Tasioula-Margari, Maria; Tsabolatidou, Eleftheria

    2015-01-01

    The aim of this study was to evaluate the recovery of individual phenolic compounds extracted from virgin olive oil (VOO), from different Greek olive varieties. Sufficient recoveries (90%) of all individual phenolic compounds were obtained using methanol as an extraction solvent, acetonitrile for residue solubilization, and two washing steps with hexane. Moreover, in order to elucidate structural characteristics of phenolic compounds in VOO, high performance liquid chromatography with a diode array detector (HPLC-DAD) at 280 and 340 nm and HPLC coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) in the negative-ion mode were performed. The most abundant phenolic compounds were oleuropein derivatives with m/z 319 and 377 and ligstroside derivatives with m/z 303, 361. Lignans, such as 1-acetoxypinoresinol and pinoresinol were also present in substantial quantities in the phenolic fraction. However, pinoresinol was co-eluted with dialdehydic form of ligstroside aglycone (DAFLA) and it was not possible to be quantified separately. The phenolic extracts, obtained from different VOO samples, yielded similar HPLC profiles. Differences, however, were observed in the last part of the chromatogram, corresponding to isomers of the aldehydic form of ligstroside aglycone. Oxidized phenolic products, originating from secoiridoids, were also detected. PMID:26783843

  16. Extraction, Separation, and Identification of Phenolic Compounds in Virgin Olive Oil by HPLC-DAD and HPLC-MS.

    PubMed

    Tasioula-Margari, Maria; Tsabolatidou, Eleftheria

    2015-01-01

    The aim of this study was to evaluate the recovery of individual phenolic compounds extracted from virgin olive oil (VOO), from different Greek olive varieties. Sufficient recoveries (90%) of all individual phenolic compounds were obtained using methanol as an extraction solvent, acetonitrile for residue solubilization, and two washing steps with hexane. Moreover, in order to elucidate structural characteristics of phenolic compounds in VOO, high performance liquid chromatography with a diode array detector (HPLC-DAD) at 280 and 340 nm and HPLC coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) in the negative-ion mode were performed. The most abundant phenolic compounds were oleuropein derivatives with m/z 319 and 377 and ligstroside derivatives with m/z 303, 361. Lignans, such as 1-acetoxypinoresinol and pinoresinol were also present in substantial quantities in the phenolic fraction. However, pinoresinol was co-eluted with dialdehydic form of ligstroside aglycone (DAFLA) and it was not possible to be quantified separately. The phenolic extracts, obtained from different VOO samples, yielded similar HPLC profiles. Differences, however, were observed in the last part of the chromatogram, corresponding to isomers of the aldehydic form of ligstroside aglycone. Oxidized phenolic products, originating from secoiridoids, were also detected. PMID:26783843

  17. Separation of colloidal two dimensional materials by density gradient ultracentrifugation

    SciTech Connect

    Kuang, Yun; Song, Sha; Huang, Jinyang; Sun, Xiaoming

    2015-04-15

    Two-dimensional (2D) materials have been made through various approaches but obtaining monodispersed simply by synthesis optimization gained little success, which highlighted the need for introducing nanoseparation methods. Density gradient ultracentrifugation method has emerged as a versatile and scalable method for sorting colloidal 2D nanomaterials. Isopycnic separation was applied on thickness-dependent separation of graphene nanosheets. And rate-zonal separation, as a more versatile separation method, demonstrated its capability in sorting nanosheets of chemically modified single layered graphene, layered double hydroxide, and even metallic Ag. Establishing such density gradient ultracentrifugation method not only achieves monodispersed nanosheets and provides new opportunities for investigation on size dependent properties of 2D materials, but also makes the surface modification possible by introducing “reaction zones” during sedimentation of the colloids. - Graphical abstract: Two-dimensional (2D) materials have been made through various approaches but obtaining monodispersed simply by synthesis optimization gained little success, which highlighted the need for introducing nanoseparation methods. Density gradient ultracentrifugation method has emerged as a versatile and scalable method for sorting colloidal 2D nanomaterials according to their size of thickness difference. Establishing such density gradient ultracentrifugation method not only achieves monodispersed nanosheets and provides new opportunities for investigation on size dependent properties of 2D materials, but also makes the surface modification possible by introducing “reaction zones” during sedimentation of the colloids. - Highlights: • Density gradient ultracentrifugation was applied on size separation of 2D material. • Isopycnic separation was applied on separation of low density materials. • Rate-zonal separation was applied on separation of large density materials. • Size

  18. HTS High Gradient Magnetic Separation system

    SciTech Connect

    Daugherty, M.A.; Coulter, J.Y.; Hults, W.L.

    1996-09-01

    We report on the assembly, characterization and operation of a high temperature superconducting (HTS) magnetic separator. The magnet is made of 624 m of Silver/BSCCO superconducting wire and has overall dimensions of 18 cm OD, 15.5 cm height and 5 cm ID. The HTS current leads are designed to operate with the warm end at 75 K and the cold end cooled by a two stage Gifford-McMahon cryocooler. The upper stage of the cryocooler cools the thermal shield and two heat pipe thermal intercepts. The lower stage of the cryocooler cools the HTS magnet and the bottom end of the HTS current leads. The HTS magnet was initially characterized in liquid cryogens. We report on the current- voltage (I-V) characteristics of the HTS magnet at temperatures ranging from 15 to 40 K. At 40 K the magnet can generate a central field of 2.0 T at a current of 120 A.

  19. Separation of aromatic carboxylic acids using quaternary ammonium salts on reversed-phase HPLC. 2. Application for the analysis of Loy Yang coal oxidation products

    SciTech Connect

    Kawamura, K.; Okuwaki, A.; Verheyen, T.V.; Perry, G.J.

    2006-07-01

    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 was optimized using the solvent gradient method. This method was applied for the analysis of Loy Yang coal oxidation products. It was confirmed that the analytical data using this method were consistent with those determined using gas chromatography.

  20. A NEW HPLC METHOD FOR SEPARATION OF PHYTOPLANKTON PIGMENTS IN NATURAL SAMPLES

    EPA Science Inventory

    A new high-performance liquid chromatographic (HPLC) method was developed to analyze, in a single run, most polar and non-polar chlorophylls and carotenoids from marine phytoplankton. The method is based on a reverse-phase amide C16 (RP-amide C16) column and an elution gradient o...

  1. SEPARATION OF OCTYLPHENOL POLYETHER ALCOHOLS SURFACTANTS BY CAPILLARY COLUMN AND HPLC

    EPA Science Inventory

    Separation of nonionic octylphenol polyether alcohols (OPA) by supercritical fluid chromatography (SFC) and HPLC is described. sing a density programming and a 50-pm i.d. capillary column, a total of 18 group oligomers was separated. he effects of the operating parameters, such a...

  2. SEPARATION OF OCTYLPHENOL POLYETHER ALCOHOLS SURFACTANTS BY CAPILLARY COLUMN SFC AND HPLC

    EPA Science Inventory

    Separation of nonionic octylphenol polyether alcohols (OPA) by supercritical fluid chromatography (SFC) and HPLC is described. Using a density programming and a 50-μm i.d. capillary column, a total of 18 group oligomers was separated. The effects of the operating parameters, such...

  3. Size-separation of silver nanoparticles using sucrose gradient centrifugation

    SciTech Connect

    Suresh, Anil K.; Pelletier, Dale A.; Moon, Ji Won; Phelps, Tommy; Doktycz, Mitchel John

    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 gradient 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.

  4. Separation and sensitive assay of THC in biological fluids by HPLC and GLC.

    PubMed

    Garrett, E R; Hunt, C A

    1976-05-01

    HPLC systems were developed to permit quantitative separation of delta9-tetrahydrocannabinol from many of the heptane extractable lipoidal and other endogenous substances in biological fluids. These substances interfered with the quantification by flame ionization GLC of unmodified compound and by electron capture GLC of pentafluorobenzoylated compound. Reverse phase HPLC elution, with 47% acetonitrile in water, and normal phase HPLC with 25% chloroform in heptane, separated tetrahydrocannabinol from 11-hydroxy-delta9-tetrahydrocannabinol and other monohydroxylated tetrahydrocannabinols. These systems also purified stock solutions of tetrahydrocannabinol from accompanying contaminants. The various monohydroxylated tetrahydrocannabinols were resolved from each other in normal phase, 80% chloroform in heptane. The delta8 and delta9-tetrahydrocannabinols were separable in normal phase with 5% tetrahydrofuran in hexane. The GLC analysis of pentafluorobenzoylated tetrahydrocannabinol had a sensitivity of 1 ng/ml of plasma with an estimated 5% standard error of an assay with the extraction and GLC procedures given herein. Radiochemical analysis of the HPLC separated fraction had s sensitivity of 0.2 ng/ml of plasma with an estimated 2% standard error of an assay. There was no significant difference between the liquid scintillation and electron capture GLC assays of the HPLC separated delta9-tetrahydrocannabinol obtained from the plasma of dogs administered the drug. Radiolabelled compounds can be added to plasma samples as internal standards to determine the recovery efficiencies of the several procedures in the analysis of unlabelled tetrahydrocannabinol. PMID:967239

  5. A gradient based facile HPLC method for simultaneous estimation of antioxidants extracted from tea powder.

    PubMed

    Nanjegowda, Shankara H; Papanna, Manasa G; Achar, Raghu Ram; Rangappa, Kanchugarakoppal S; Mallu, Puttaswamappa; Swamy, Shivananju Nanjunda

    2016-05-01

    A new simple, rapid and precise RP-HPLC method was developed for the extraction and quantitative estimation of caffeine (C), (-)-epigallocatechin gallate (EGCG), (+)-catechin(Ct), (-)-epicatechin(EC), and (-)-epicatechin gallate (ECG) (collectively named as Tea Powder Bioactives TPBAs) extracted from tea powder using different ratios of ethanol: water. The simultaneous determination of TPBAs was performed using the UV spectrophotometric method which employs the absorbance at 205 nm (λmax of caffeine and polyphenols). This method is a gradient based HPLC method with a flow rate of 0.8 mL/min using Inertsil ODS 100 × 4.6 mm, 3 μm column with methanol and ammonium dihydrogen phosphate (pH-2.8) as mobile phase. The method was validated in terms of specificity, precision, linearity, accuracy, limit of quantification (LOQ), and limit of detection (LOD). The linearity of the proposed method was investigated for concentration ranging between 0.5-60 μg/mL with regression co-efficient, R(2) = 0.999-1.0. This method estimates all the TPBAs simultaneously with enhanced precision and linearity as per the ICH guidelines. Also, to confirm the individual TPBA, the antioxidant property of the each TPBA was analyzed which was commensurate with that of the previous reports. PMID:27407191

  6. Separation of colloidal two dimensional materials by density gradient ultracentrifugation

    NASA Astrophysics Data System (ADS)

    Kuang, Yun; Song, Sha; Huang, Jinyang; Sun, Xiaoming

    2015-04-01

    Two-dimensional (2D) materials have been made through various approaches but obtaining monodispersed simply by synthesis optimization gained little success, which highlighted the need for introducing nanoseparation methods. Density gradient ultracentrifugation method has emerged as a versatile and scalable method for sorting colloidal 2D nanomaterials. Isopycnic separation was applied on thickness-dependent separation of graphene nanosheets. And rate-zonal separation, as a more versatile separation method, demonstrated its capability in sorting nanosheets of chemically modified single layered graphene, layered double hydroxide, and even metallic Ag. Establishing such density gradient ultracentrifugation method not only achieves monodispersed nanosheets and provides new opportunities for investigation on size dependent properties of 2D materials, but also makes the surface modification possible by introducing "reaction zones" during sedimentation of the colloids.

  7. MAGNETITE RECOVERY IN COAL WASHING BY HIGH GRADIENT MAGNETIC SEPARATION

    EPA Science Inventory

    The report describes a demonstration of the successful recovery of magnetite from mixtures of magnetite and coal, like those found in a coal-washing circuit, by High Gradient Magnetic Separation. The demonstration was part of a research program at Francis Bitter National Magnet L...

  8. TREATMENT OF COMBINED SEWER OVERFLOWS BY HIGH GRADIENT MAGNETIC SEPARATION

    EPA Science Inventory

    Seeded water treatment by high gradient magnetic separation techniques was carried out on combined storm overflows and raw sewage influents. Both bench-type and continuous pilot plant tests were performed to evaluate the effectiveness of the process in purifying waste waters. Cri...

  9. Development of high gradient magnetic separation system under dry condition

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

    The interfusion of impurities such as metallic wear debris has been a problem in the manufacturing process of foods, medicines, and industrial products. Gravity separation system and membrane separation system has been used widely for powder separation, however magnetic separation system is much efficient to separate magnetic particles. Magnetic separation system under wet process is used conventionally, however, it has some demerit such as necessity of drying treatment after separation and difficulty of running the system in the cold region and so on. Thus, magnetic separation under dry process is prospective as alternative method. In this paper, we developed high gradient magnetic separation system (HGMS) under dry process. In dry HGMS system, powder coagulation caused by particle interaction is considerable. Powder coagulation causes a blockage of magnetic filters and results in decrease of separation performance of dry HGMS system. In order to investigate the effect of powder coagulation on separation performance, we conducted experiments with two kinds of powdered materials whose cohesive properties are different.

  10. Size-separation of silver nanoparticles using sucrose gradient centrifugation

    DOE PAGESBeta

    Suresh, Anil K.; Pelletier, Dale A.; Moon, Ji Won; Phelps, Tommy; Doktycz, Mitchel John

    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

  11. Characterization of zebrafish cardiac proteome using online pH gradient SCX-RP HPLC-MS/MS platform.

    PubMed

    Zhang, Jiang; Lanham, Kevin A; Heideman, Warren; Peterson, Richard E; Li, Lingjun

    2013-01-01

    Two-dimensional HPLC coupled with tandem MS (MS/MS) has become a mainstream technique in the shotgun proteomics for large-scale identification of proteins from biological samples. This powerful technology provides speed, sensitivity, and dynamic range which are essential to probe complex peptide mixtures from proteomic samples. Herein we present a pH gradient SCX-RP 2D HPLC-MS/MS method designed to improve the peptide resolution and protein identification from complex proteomic samples. The comparison between the pH gradient SCX-RP 2D HPLC method and traditional salt gradient SCX-RP method was presented. A two-step sample prefractionation method utilizing microwave-assisted tryptic digestion to improve the identification of insoluble proteins was also introduced. This novel 2D HPLC-MS/MS method was applied to the heart proteomic sample of the zebrafish, Danio rerio, to provide comprehensive cardiac proteomic profiling of this important model organism for cardiovascular and environmental toxicology studies. PMID:23606253

  12. Liquid mixture convection during phase separation in a temperature gradient

    NASA Astrophysics Data System (ADS)

    Lamorgese, A. G.; Mauri, R.

    2011-03-01

    We simulate the phase separation of a low-viscosity binary mixture, assuming that the fluid system is confined between two walls that are cooled down to different temperatures below the critical point of the mixture, corresponding to quenches within the unstable range of its phase diagram. Spinodal decomposition patterns for off-critical mixtures are studied numerically in two dimensions in the creeping flow limit and for a large Lewis number, together with their dependence on the fluidity coefficient. Our numerical results reproduce the large-scale unidirectional migration of phase-separating droplets that was observed experimentally by Califano et al. ["Large-scale, unidirectional convection during phase separation of a density-matched liquid mixture," Phys. Fluids 17, 094109 (2005)], who measured typical speeds that are quite larger than the Marangoni velocity. To understand this finding, we then studied the temperature-gradient-induced motion of an isolated droplet of the minority phase embedded in a continuous phase, showing that when the drop is near local equilibrium, its speed is of the same order as the Marangoni velocity, i.e., it is proportional to the unperturbed temperature gradient and the fluidity coefficient. However, far from local equilibrium, i.e., for very large unperturbed temperature gradients, the drop first accelerates to a speed that is larger than the Marangoni velocity, then, later, it decelerates, exhibiting an increase-decrease behavior, as described by Yin et al. ["Thermocapillary migration of nondeformable drops," Phys. Fluids 20, 082101 (2008)]. Such behavior is due to the large nonequilibrium, Korteweg-driven convection, which at first accelerates the droplets to relatively large velocities, and then tends to induce an approximately uniform inside temperature distribution so that the drop experiences an effective temperature gradient that is much smaller than the unperturbed one and, consequently, decelerates.

  13. Microchannel protein separation by electric field gradient focusing.

    PubMed

    Petsev, Dimiter N; Lopez, Gabriel P; Ivory, Cornelius F; Sibbett, Scott S

    2005-06-01

    A microchannel device is presented which separates and focuses charged proteins based on electric field gradient focusing. Separation is achieved by setting a constant electroosmotic flow velocity against step changes in electrophoretic velocity. Where these two velocities are balanced for a given analyte, the analyte focuses at that point because it is driven to it from all points within the channel. We demonstrate the separation and focusing of a binary mixture of bovine serum albumin and phycoerythrin. The device is constructed of intersecting microchannels in poly(dimethylsiloxane)(PDMS) inlaid with hollow dialysis fibers. The device uses no exotic chemicals such as antibodies or synthetic ampholytes, but operates instead by purely physical means involving the independent manipulation of electrophoretic and electroosmotic velocities. One important difference between this apparatus and most other devices designed for field-gradient focusing is the injection of current at discrete intersections in the channel rather than continuously along the length of a membrane-bound separation channel. PMID:15915250

  14. 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. PMID:27294532

  15. Identification and quantification of phenolic compounds in grapes by HPLC-PDA-ESI-MS on a semimicro separation scale.

    PubMed

    Nicoletti, Isabella; Bello, Cristiano; De Rossi, Antonella; Corradini, Danilo

    2008-10-01

    Reversed phase high performance liquid chromatography (RP-HPLC) on a semimicro separation scale was employed to develop a straightforward method for the simultaneous separation, identification, and quantification of phenolic compounds occurring in whole berries of Vitis vinifera, which comprise phenolic acids, flavonols, catechins, stilbenes, and anthocyanins. A C-18 narrow bore column of 150 x 2.0 mm I.D. and a semimicro photodiode array detector (PDA) cell of 2.5 microL, in conjunction with a mass spectrometry detector equipped with an electrospray ionization source (ESI-MS) to confirm peak identification, were employed. The C-18 narrow bore column was eluted by a multisegment gradient of increasing concentration of acetonitrile in water-formic acid solution that was optimized on the basis of the results of a study carried out to evaluate the influence of mobile phase composition and gradient shape on separation performance and detection sensitivity by ESI-MS. The identification of individual phenolic compounds was performed on the basis of their retention times and both UV-visible and mass spectra, acquired by a mass spectrometer (MS) equipped with an electrospray ionization (ESI) source, employed in conjunction with the PDA detector. Libraries comprising retention times, UV-visible, and mass spectra for major phenolic compounds expected in grape berries were made by subjecting solutions of each phenolic standard to the optimized RP-HPLC method. Quantification of individual compounds was performed by the external standard method using a six point regression graph of the UV-visible absorption data collected at the wavelength of maximum absorbance of each analyte determined by the PDA spectra. The RP-HPLC method was validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability, and accuracy, which was evaluated by a recovery study. The developed method was successfully applied to identify the phenolic compounds

  16. Investigation of open-gradient magnetic separation for Illinois coal

    SciTech Connect

    Doctor, R.D.; Livengood, C.D.; Genens, L.E.; Swietlik, C.E.; Foote, K.

    1987-01-01

    Open-gradient magnetic separation (OGMS) using superconducting quadrupole magnets is a novel coal-beneficiation technology offering high pyritic-sulfur removal from pulverized dry coal. The system operates in a continuous mode, uses no chemicals, and has an estimated power demand 75% lower than techniques using conventional electromagnets, while achieving magnetic separation forces up to 267% higher. Specifically applicable to finely ground coal (120 to 325 mesh), OGMS could encourage the commercialization of other developing coal technologies, such as coal-water slurries, fludized-bed combustion, and coal synfuels. Both the experimental program conducted by Argonne National Laboratory and the results of modeling in support of the experimental program are described. 11 refs., 9 figs.

  17. Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications

    PubMed Central

    Haverick, Mark; Mengisen, Selina; Shameem, Mohammed; Ambrogelly, Alexandre

    2014-01-01

    Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) is a powerful analytical method used for the separation of molecular variants of therapeutic proteins. The method has been employed for monitoring various post-translational modifications, including proteolytic fragments and domain misfolding in etanercept (Enbrel®); tryptophan oxidation, aspartic acid isomerization, the formation of cyclic imide, and α amidated carboxy terminus in recombinant therapeutic monoclonal antibodies; and carboxy terminal heterogeneity and serine fucosylation in Fc and Fab fragments. HIC-HPLC is also a powerful analytical technique for the analysis of antibody-drug conjugates. Most current analytical columns, methods, and applications are described, and critical method parameters and suitability for operation in regulated environment are discussed, in this review. PMID:24751784

  18. 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

  19. Differential blood cell separation using a high gradient magnetic field.

    PubMed

    Paul, F; Roath, S; Melville, D

    1978-02-01

    A technique for the separation of erythrocytes from whole blood is described which exploits the magnetic property of haemoglobin in the reduced state. The technique is characterized by the use of a filter consisting of a cylinder, containing stainless steel wire mesh, placed between the jaws of an electro magnet. When activated, the electromagnet induces a magnetic field gradient in the vicinity of each of the constituent wires, sufficient to attract and trap erythrocytes in suspension. The number of erythrocytes captured varies with the applied field (0-1.4 Tesla in these experiments) and flow rate (1.9-12.9 x 10(-4) m s-1). The capture process does not cause haemolysis or observable surface damage to the erythrocytes and neither leucocytes nor platelets are retained by the filter. PMID:638075

  20. Modeling high gradient magnetic separation from biological fluids.

    SciTech Connect

    Bockenfeld, D.; Chen, H.; Rempfer, D.; Kaminski, M. D.; Rosengart, A. J.; Chemical Engineering; Illinois Inst. of Tech.; Univ. of Chicago, Pritzker School of Medicine

    2006-01-01

    A proposed portable magnetic separator consists of an array of biocompatible capillary tubing and magnetizable wires immersed in an externally applied homogeneous magnetic field. While subject to the homogeneous magnetic field, the wires create high magnetic field gradients, which aid in the collection of blood-borne magnetic nanospheres from blood flow. In this study, a 3-D numerical model was created using COMSOL Multiphysics 3.2 software to determine the configuration of the wire-tubing array from two possible configurations, one being an array with rows alternating between wires and tubing, and the other being an array where wire and tubing alternate in two directions. The results demonstrated that the second configuration would actually capture more of the magnetic spheres. Experimental data obtained by our group support this numerical result.

  1. Microfluidic Precolumn Derivatization of Environmental Phenols with Coumarin-6-Sulfonyl Chloride and HPLC Separation.

    PubMed

    Suliman, FakhrEldin O; Al-Busaidi, Jihad N; Al-Lawati, Hiader A J; Al-Kindy, Salma M Z

    2015-09-01

    A simple, fast, sensitive and versatile method for the analysis of phenols in water is proposed using microfluidic precolumn derivatization with the fluorogenic label coumarin-6-sulfonyl chloride (C6SCl) and HPLC separation on monolithic columns. Phenols react with C6SCl within 3.0 min in the microreactor at ambient temperature to produce phenol-coumarin sulphonamides derivatives which were separated in reversed phase high-performance liquid chromatography followed by postcolumn ring-opening and fluorescence detection at λexc = 360 nm and λem = 460 nm. The optimum conditions for the derivatization, separation and ring-opening reaction have been established. The calibration curves were linear for the studied phenols in the range of 0.75-12.5 mg L(-1). The application of the method to environmental samples was demonstrated by analyzing tap and fountain water samples spiked with the phenolic compounds. PMID:25809998

  2. Capture of metallic copper by high gradient magnetic separation system.

    PubMed

    Wu, Wan-I; Wu, Chung-Hsin; Hong, P K Andy; Lin, Cheng-Fang

    2011-10-01

    Valence copper was recovered from wastewater by chemical reduction and use of a high gradient magnetic separation (HGMS) system. Ammonia (NH3) and sodium dithionate (Na2S2O4) at a molar ratio of [Cu]:[NH3]:[Na2S2O4] = 1:4:3 at pH = 9.5 were used first to chemically reduce copper ion to metallic copper; the resultant metal solids were captured in an upflowing reactor space equipped with a permalloy matrix net under a high gradient magnetic field. The captured solids were predominantly 6-20 microm in diameter, with Cu2O and CuO present among the solids. Four treatment configurations with and without the use of magnetic field and metal alloy as the matrix net were tested and their effects evaluated: (1) no magnetic field or matrix, (2) no magnetic field but with matrix, (3) with magnetic field but no matrix, (4) with both magnetic field and matrix. At flow rates of 40, 60, 80 and 100 cm3/min, capture efficiencies for metallic copper in the absence of magnetic field were 87%, 86%, 63%, and 39%, respectively, and in the presence of magnetic field were 99%, 98%, 95%, and 93%, respectively. The HGMS was critical for a high capture efficiency, whereas a matrix net only marginally enhanced it. Additional tests with a larger reactor confirmed similarly high efficiencies of > 85%. The use of an alloy matrix appeared to be important when high flow rates are most likely to be employed in practical applications. PMID:22329132

  3. Feasibility study of iron mineral separation from red mud by high gradient superconducting magnetic separation

    NASA Astrophysics Data System (ADS)

    Li, Yiran; Wang, Jun; Wang, Xiaojun; Wang, Baoqiang; Luan, Zhaokun

    2011-02-01

    The disposal of bayer red mud tailings now seriously threats the environment safety. Reduction and recycling of red mud is now an urgent work in aluminum industry. High gradient superconducting magnetic separation (HGSMS) system was applied to separate the extreme fine RM particles (<100 μm) into high iron content part and low iron content part. Two sorts of RM were fed in the HGSMS. The iron oxide contents in concentrates were about 65% and 45% when RM 1# and RM 2# were fed respectively. Meanwhile, the residues contained 52.0% or 14.1% iron oxide in residues after eight separation stages when RM 1# and RM 2# were fed respectively. The mass recovery of iron concentrates was about 10% after once separation process regardless of RM 1# or RM 2# was fed. Extreme fine particles (<10 μm) could be captured in the HGSMS. Intergrowth of Fe and other elements is disadvantages for iron mineral separation from RM by HGSMS. Some improvement should be studied to enhance the efficiency of iron separation. It is possible for HGSMS to separate RM into high iron content part and low iron content part, the former part could be used in iron-making furnace and the later part could be recycling to sintering process for alumina production or used as construction material.

  4. High-capacity stationary phases containing heavy atoms for HPLC separation of fullerenes

    SciTech Connect

    Kimata, Kazuhiro |; Hirose, Tsunehisa; Moriuchi, Kouji; Hosoya, Ken; Araki, Takeo; Tanaka, Nobuo

    1995-08-01

    A high-capacity stationary phase for the separation of fullerenes was prepared by immobilizing 3-[(pentabromobenzyl)oxy]propylsilyl (PBB) groups onto silica surfaces. The stationary phase was developed by a reciprocal approach. This was possible by finding the structure of solvents that provided high solubilities as well as high eluent strength for chromatographic elution of fullerenes. The increased solubility and increased eluent strength for C{sub 60} seen with solvents containing heavy heteroatoms suggested the preferential interaction of C{sub 60} with such solvent molecules. The stationary phases containing sulfur, chlorine, or bromine in fact resulted in longer retention of fullerenes. The PBB silica showed high retentivity with excellent efficiency for fullerenes, permitting the use of solvents providing high solubilities, such as carbon disulfide and 1,2,4-trichlorobenzene for gramscale separations with ordinary HPLC equipment. 22 refs., 6 figs., 3 tabs.

  5. Investigation of the separation of scandium and rare earth elements from red mud by use of reversed-phase HPLC.

    PubMed

    Tsakanika, Lambrini V; Ochsenkühn-Petropoulou, Maria Th; Mendrinos, Leonidas N

    2004-07-01

    A chromatographic method has been developed for separation and determination of scandium (Sc) and rare earth elements (REEs) in samples from a red mud (RM)-utilization process. Reversed-phase high-performance liquid chromatography (RP-HPLC) with post-column derivatization using 4-(2-pyridylazo)-resorcinol (PAR) and UV-visible detection at 520 nm was tested using different gradient elution profiles and pH values to optimize separation and recovery, primarily for Sc but also for yttrium and the individual lanthanides, from iron present in the samples. The separation was performed in less than 20 min by use of a mobile phase gradient. The concentration of alpha-hydroxyisobutyric acid ( alpha-HIBA), as eluent, was altered from 0.06 to 0.4 mol L(-1) (pH 3.7) and 0.01 mol L(-1) sodium salt n-octane sulfonic acid (OS) was used as modifier. Very low detection limits in the nanogram range and a good resolution for Sc and REEs except for Y/Dy were achieved. Before application of the method to the red mud samples and to the corresponding bauxites, Sc and REEs were leached from red mud with 0.6 mol L(-1) HNO(3) and mostly separated, as a group, from the main elements by ion exchange/selective elution (6 mol L(-1) HNO(3)) in accordance with a pilot-plant process developed in this laboratory. After evaporation of the eluent to dryness the extracted elements were re-dissolved in the mobile phase. By use of this chromatographic method Sc, which is the most expensive of the elements investigated and occurs in economically interesting concentrations in red mud, could be separated not only from co-existing Fe but also from Y/Dy, Yb, Er, Ho, Gd, Eu, Sm, Nd, Pr, Ce and La. All the elements investigated were individually recovered. Their recoveries were found to be nearly quantitative. PMID:15221192

  6. 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. PMID:21316945

  7. Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions.

    PubMed

    Gilar, Martin; Olivova, Petra; Daly, Amy E; Gebler, John C

    2005-09-01

    Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis. PMID:16224963

  8. 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

  9. Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC.

    PubMed

    Borrego, C M; Garcia-Gil, L J

    1994-07-01

    A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and β-carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status. PMID:24310022

  10. High-gradient magnetic separation using ferromagnetic membrane

    NASA Astrophysics Data System (ADS)

    Podoynitsyn, Sergey N.; Sorokina, Olga N.; Kovarski, Alexander L.

    2016-01-01

    The magnetic separator with the membrane separating unit made of laser-perforated thick ferromagnetic foil was tested using composite water suspension of magnetic nanoparticles adsorbed on hydroxylapatite microparticles. The average sizes of the particles in the suspension and the magnetic moment of the suspension were measured by dynamic light scattering and electron magnetic resonance correspondingly to evaluate the efficiency of the separation. It was shown experimentally that the separation is effected by the membrane type and the flow rate. Magnetic coarse grains (larger than 1 μm) were captured by the membrane preferably and the magnetic moment of the suspension decreased by 20-25% after the separation. The magnetic field simulation and experimental results demonstrate the higher separation efficiency for thicker membranes.

  11. Separated flows developing under increasingly adverse pressure gradients

    NASA Technical Reports Server (NTRS)

    Mehta, Jayesh M.

    1989-01-01

    Experimental results are presented for separating flows over a NASA GA(W)-1 airfoil with 2-percent trailing-edge thickness, with emphasis on the mean velocity data that demonstrate flowfield similarity downstream of separation. The length scale presently employed was selected because it furnishes the requisite matching point between the outer, mixing region flow and the back-flow.

  12. Separation and identification of oligomeric vinylmethoxysiloxanes by gradient elution chromatography coupled with electrospray ionization mass spectrometry.

    PubMed

    Jia, Guiying; Wan, Qian-Hong

    2015-05-22

    A high-performance liquid chromatography with online electrospray ionization mass spectrometry (HPLC-ESI-MS) has been used to separate and identify the reaction products resulting from controlled acid-catalyzed hydrolytic polycondensation of vinyltrimethoxysilane (VMS). The reaction products were prepared in the molar ratio of water to VMS (r1) ranging from 0.6 to 1.2, characterized by standard spectroscopic techniques, and subsequently analyzed by HPLC-UV absorbance detection and HPLC-ESI-MS. Linear vinylmethoxysiloxane oligomers with the number of repeat units (n) ranging from 3 to 11 are predominant species at the beginning of the reaction (for r1=0.6). Then they transform into monocyclic (for r1=1.0) and bicyclic (for r1=1.2) species with gradually increasing amount of water in the reaction mixture. The oligomer conversions suggest that structure growth of vinylmetoxysiloxanes proceeds by nonrandom cyclization reactions, which are favored over chain extension under the chosen reaction conditions. Direct ESI-MS, HPLC-ESI-MS and HPLC-UV were used to determine the molar mass distributions for the vinylmethoxysiloxane oligomers prepared in three different values of r1. The molar mass averages increase slightly with the amount of water in the reaction mixture and vary somewhat with the method used. Our results indicate that with the combined capability of separation, sensitivity and identification, HPLC-ESI-MS is especially useful to study highly complex silicon-based compounds with hyperbranched, caged or cubic structures as building blocks for hybrid materials. PMID:25890439

  13. 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. PMID:26838399

  14. Determination of arsenic species in edible periwinkles (Littorina littorea) by HPLC-ICPMS and XAS along a contamination gradient.

    PubMed

    Whaley-Martin, K J; Koch, I; Reimer, K J

    2013-07-01

    Arsenic is naturally found in the tissues of marine animals, usually as the non-toxic arsenical arsenobetaine, but exposure to elevated arsenic concentrations in the environment may alter the arsenic species distribution within tissues of the organism. This study examined the arsenic species in the tissues of the marine periwinkle (Littorina littorea) along an arsenic concentration gradient in the sediment. The arsenicals in L. littorea were examined using the complementary analytical methods high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICPMS) and X-ray absorption spectroscopy (XAS). Total arsenic concentrations in the periwinkle tissues ranged from 56 to 840 mg·kg(-1) dry weight (equivalent to 13 to 190 mg·kg(-1) wet weight). Inorganic arsenicals were found to be positively correlated with total arsenic concentrations (R(2)=0.993) and reached 600 mg·kg(-1) dry weight, the highest reported to date in marine organisms. These high inorganic arsenic concentrations within this low trophic organism pose a potential toxicological risk to higher trophic consumers. PMID:23588137

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

    SciTech Connect

    Slater, Michael; Snauko, Marian; Svec, Frantisek; Frechet, JeanM.J.

    2006-01-02

    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 the 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.

  16. Rapid separation of Arabidopsis male gametophyte developmental stages using a Percoll gradient.

    PubMed

    Dupl'áková, Nikoleta; Dobrev, Petre I; Reňák, David; Honys, David

    2016-10-01

    Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation. PMID:27583643

  17. Separation of rat tissue histone H1 subtypes by reverse-phase h.p.l.c. Identification and assignment to a standard H1 nomenclature.

    PubMed

    Lindner, H; Helliger, W; Puschendorf, B

    1990-07-15

    H1 histones from rat liver and rat testis were separated by reverse-phase h.p.l.c. Within 40 min six subfractions (H1(0), H1b, H1a, H1d, H1e + H1c and H1c) and seven subfractions (H1(0), H1b, H1a, H1d, H1e + H1c, H1c and H1t) respectively were isolated by using a linear acetonitrile gradient. Each individual H1 subtype was identified either by comparing the H1 variants (contained in both tissues but in different quantities) or by SDS/PAGE and acetic acid/urea/PAGE. Moreover, all H1 variants were characterized by amino acid analyses. The amino acid compositions of rat histone subfractions H1(0), H1b and H1e were determined for the first time. It was possible to classify unambiguously the H1 subfractions obtained by h.p.l.c. by following the standardized H1 nomenclature for electrophoretic systems recommended by Lennox, Oshima & Cohen [(1982) J. Biol. Chem. 257, 5183-5189]. Incorrect assignments that have been made in various publications are discussed. PMID:2386482

  18. Cytochrome P450 bio-affinity detection coupled to gradient HPLC: on-line screening of affinities to cytochrome P4501A2 and 2D6.

    PubMed

    Kool, Jeroen; van Liempd, Sebastiaan M; Harmsen, Stefan; Beckman, Joran; van Elswijk, Danny; Commandeur, Jan N M; Irth, Hubertus; Vermeulen, Nico P E

    2007-10-15

    Here we describe novel on-line human CYP1A2 and CYP2D6 Enzyme Affinity Detection (EAD) systems coupled to gradient HPLC. The use of the systems lies in the detection of individual inhibitory ligands in mixtures (e.g. metabolic mixtures or herbal extracts) towards two relevant drug metabolizing human CYPs. The systems can rapidly detect individual compounds in mixtures with affinities to CYP1A2 or 2D6. The HPLC-EAD systems were first evaluated and validated in flow injection analysis mode. IC50 values of known ligands for both CYPs, tested both in flow injection and in HPLC mode, were well comparable with those measured in microplate reader formats. Both EAD systems were also connected to gradient HPLC and used to screen known compound mixtures for the presence of CYP1A2 and 2D6 inhibitors. Finally, the on-line CYP2D6 EAD system was used to screen for the inhibitory activities of stereoisomers of a mixture of five methylenedioxy-alkylamphetamines (XTC analogs) on a chiral analytical column. PMID:17826363

  19. 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. PMID:24316526

  20. Separating natural and contaminant related gradients in estuarine macrobenthic community structure

    SciTech Connect

    Rakocinski, C.; Heard, R.; Walker, W.; Brown, S.; Gaston, G.; Summers, J.K.

    1995-12-31

    Using whole-community macrobenthic responses to assess pollution impacts in estuaries presents a difficult challenge due to dynamic natural conditions that may impose their own physical limitations on the biota. For example, the recognition of bioindicator taxa becomes confounded when correlations exist between gradients in natural environmental variables, such as salinity, and gradients in contaminants, such as trace metals. The authors used partial Canonical Correspondence Analysis (CCA) to separate natural and contaminant related gradients in macrobenthic community structure across 319 EMAP sites dispersed throughout the northern Gulf of Mexico. Residual variation in macrobenthic community structure was examined with respect to gradients in contaminant levels to identify responses by positive and negative bioindicator taxa. Gradients in concentrations of trace metals do not coincide completely with those in other chemical contaminants, and responses by characteristic bioindicator taxa reveal information regarding effects of specific contaminants. Several indigenous taxa serve as good negative bioindicators, whereas other opportunistic taxa serve as positive bioindicators of estuarine contamination.

  1. 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

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

    PubMed Central

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

    2014-01-01

    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 1H NMR. PMID:25745338

  3. Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography

    PubMed Central

    Wang, Xiaoli; Stoll, Dwight R; Schellinger, Adam P; Carr, Peter W.

    2008-01-01

    The optimization of peak capacity in gradient elution RPLC is essential for the separation of multi-component samples such as those encountered in proteomic research. In this work we study the effect of gradient time (tG), flow rate (F), temperature (T) and final eluent strength (ϕfinal) on the peak capacity of separations of peptides that are representative of the range in peptides found in a tryptic digest. We find that there are very strong interactions between the individual variables (e.g. flow rate and gradient time) which make the optimization quite complicated. On a given column, one should first set the gradient time to the longest tolerable and then set the temperature to the highest achievable with the instrument. Next, the flow rate should be optimized using a reasonable but arbitrary value of ϕfinal. Last, the final eluent strength should be adjusted so that the last solute elutes as close as possible to the gradient time. We also develop an easily implemented, highly efficient and effective Monte Carlo search strategy to simultaneously optimize all the variables. We find that gradient steepness is an important parameter that influences peak capacity and an optimum range of gradient steepness exists in which the peak capacity is maximized. PMID:16689544

  4. Non-HPLC rapid separation of metallofullerenes and empty cages with TiCl4 Lewis acid.

    PubMed

    Akiyama, Kazuhiko; Hamano, Tatsuyuki; Nakanishi, Yusuke; Takeuchi, Erina; Noda, Shoko; Wang, Zhiyong; Kubuki, Shiro; Shinohara, Hisanori

    2012-06-13

    Rapid and efficient separation/purification of pure metallofullerenes M(x)@C(n) (M = metal; x = 1, 2; n > 70) and carbide metallofullerenes of the type M(y)C(2)@C(n-2) (y = 2, 3, 4; n - 2 > 68) has been reported. The present method utilizes rapid and almost perfect preferential formation of TiCl(4) (generally known as a Lewis acid)-metallofullerene complexes, which easily decompose to provide pure metallofullerene powders by a simple water treatment. The present method enables one to separate the metallofullerenes up to >99% purity within 10 min without using any type of high-performance liquid chromatography (HPLC). It is found that the oxidation potentials of the metallofullerenes are crucial factors for efficient purification. The current separation/purification technique may open a brand-new era for inducing further applications and commercialization of endohedral metallofullerenes. PMID:22591414

  5. Chiral HPLC Separation and Modeling of Four Stereomers of DL-Leucine-DL-Tryptophan Dipeptide on Amylose Chiral Column.

    PubMed

    Alajmi, Mohammed F; Hussain, Afzal; Suhail, Mohd; Mukhtar, Sofi Danish; Sahoo, Dibya Ranjan; Asnin, Leonid; Ali, Imran

    2016-09-01

    Chiral high-performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL-leucine-tryptophan DL-dipeptide on AmyCoat-RP column are described. The mobile phase applied was ammonium acetate (10 mM)-methanol-acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL-, DD-, DL-, and LD- stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0-2.3 and 5.6-14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π-π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL-leucine-DL-tryptophan in unknown matrices. Chirality 28:642-648, 2016. © 2016 Wiley Periodicals, Inc. PMID:27474783

  6. Effect of combined density gradient centrifugation on X- and Y- sperm separation and chromatin integrity

    PubMed Central

    Esmaeilpour, Tahereh; Elyasi, Leila; Bahmanpour, Soghra; Ghannadi, Alireza; Monabbati, Ahmad; Dehghani, Farzaneh; Kazerooni, Marjaneh

    2012-01-01

    Background: It has been claimed that by using different washing methods, the sperms can be separated according to size, motility, density, chromosomal content and surface markings and charge. These methods also reduce sperm chromatin deficiencies and screen the sperms before applying in assisted reproduction techniques. Objective: This study compared simple density gradient methods and a combined method with albumin density gradient and PureSperm separation (alb/PureSperm) for sex preselection by double fluorescence in situ hybridization (FISH) versus chromomycin A3 staining to determine chromatin integrity. Materials and Methods: 30 normal semen samples were prepared with PureSperm, albumin gradients and alb/PureSperm. All samples were then stained by FISH and chromomycin A3. The results were compared with SPSS 11.5 and the Kruskal-Wallis test. Results: The proportion of X-bearing spermatozoa by PureSperm separation (47.58±5.67) and Y-bearing spermatozoa by albumin gradient (46.13±3.83) methods were slightly higher than in putative normal sperm samples (1:1), but there were no significant differences in the X- or Y- bearing spermatozoa counts among the three methods. Albumin gradient separation tended to underestimate abnormal spermatozoa compared to PureSperm and combined alb/PureSperm. Conclusion: Routine separation methods slightly enriched X- or Y- bearing spermatozoa, but the differences were not significant for clinical purposes. The combined alb/PureSperm method had no advantages for assessing sex ratio or chromatin integrity compared to simpler gradient methods. PMID:25246909

  7. Turbulence characteristics of separated boundary layer flow under unsteady pressure gradients using direct numerical simulation

    NASA Astrophysics Data System (ADS)

    Park, Junshin; Bromby, William; You, Donghyun

    2013-11-01

    To understand turbulence characteristics of separated boundary layer flow under unsteady pressure gradients, a direct numerical simulation study is performed. Steady and unsteady blowing-suction velocity distributions are imposed along the upper boundary of the computational domain to introduce steady and unsteady adverse pressure gradients leading to steady and unsteady separated turbulent boundary layers, respectively. Time averaged and phase averaged turbulence statistics such as velocity, vorticity, kinetic energy budgets, Reynolds stress budgets, wall pressure fluctuations and skin friction distributions are examined in detail with aims of gaining understanding of flow physics for unsteady separated turbulent boundary layer and the sources of incapability of the conventional Reynolds-averaged Navier-Stokes models in predicting unsteady separation. Supported by the Army Research Office Grant W911NF1010348 and the National Research Foundation of Korea Grant NRF-2012R1A1A2003699.

  8. Harvesting fresh water and marine algae by magnetic separation: screening of separation parameters and high gradient magnetic filtration.

    PubMed

    Cerff, Martin; Morweiser, Michael; Dillschneider, Robert; Michel, Aymeé; Menzel, Katharina; Posten, Clemens

    2012-08-01

    In this study, the focus is on magnetic separation of fresh water algae Chlamydomonas reinhardtii and Chlorella vulgaris as well as marine algae Phaeodactylum tricornutum and Nannochloropsis salina by means of silica-coated magnetic particles. Due to their small size and low biomass concentrations, harvesting algae by conventional methods is often inefficient and cost-consuming. Magnetic separation is a powerful tool to capture algae by adsorption to submicron-sized magnetic particles. Hereby, separation efficiency depends on parameters such as particle concentration, pH and medium composition. Separation efficiencies of >95% were obtained for all algae while maximum particle loads of 30 and 77 g/g were measured for C. reinhardtii and P. tricornutum at pH 8 and 12, respectively. This study highlights the potential of silica-coated magnetic particles for the removal of fresh water and marine algae by high gradient magnetic filtration and provides critical discussion on future improvements. PMID:22705536

  9. Application of high temperature superconductors to high-gradient magnetic separation

    SciTech Connect

    Daugherty, M.A.; Prenger, F.C.; Hill, D.D.; Daney, D.E.; Worl, L.W.; Schake, A.R.; Padilla, D.D.

    1994-06-01

    High Gradient Magnetic Separation (HGMS) is a powerful technique which can be used to separate widely dispersed contaminants from a host material, This technology can separate magnetic solids from other solids, liquids or gases. As the name implies HGMS uses large magnetic field gradients to separate ferromagnetic and paramagnetic particles. HGMS separators usually consist of a high-field solenoid magnet, the bore of which contains a fine-structured, ferromagnetic matrix material. The matrix material locally distorts the magnetic field and creates large field gradients in the vicinity of the matrix elements. These elements then become trapping sites for magnetic particles and are the basis for the magnetic separation. In this paper we discuss the design and construction of a prototype HGMS unit using a magnet made with high temperature superconductors (HTS). The prototype consists of an outer vacuum vessel which contains the HTS solenoid magnet The magnet is surrounded by a thermal radiation shield and multilayer insulation (MLI) blankets. The magnet, thermal shield and current leads all operate in a vacuum and are cooled by a cryocooler. High temperature superconducting current leads are used to reduce the heat leak from the ambient environment to the HTS magnet.

  10. Magnetic force on a magnetic particle within a high gradient magnetic separator

    NASA Astrophysics Data System (ADS)

    Baik, S. K.; Ha, D. W.; Kwon, J. M.; Lee, Y. J.; Ko, R. K.

    2013-01-01

    HGMS (High Gradient Magnetic Separator) uses matrix to make high magnetic field gradient so that ferro- or para-magnetic particles can be attracted to them by high magnetic force. The magnetic force generated by the field gradient is several thousand times larger than that by background magnetic field alone. So the HGMS shows excellent performance compared with other magnetic separators. These matrixes are usually composed of stainless steel wires having high magnetization characteristics. This paper deals with superconducting HGMS which is aimed for purifying waste water by using stainless steel matrix. Background magnetic field up to 6 T is generated by a superconducting solenoid and the stainless steel matrixes are arranged inside of the solenoid. Based on magnetic field calculated by FEM (Finite Element Method), we could calculate magnetic force acting on a magnetic particle such as hematite and maghemite consisting of major impurities in the condenser water of a thermal power station.

  11. APPLICATION OF HIGH-GRADIENT MAGNETIC SEPARATION TO FINE PARTICLE CONTROL

    EPA Science Inventory

    The report gives results of an assessment of the potential use of high-gradient magnetic separation (HGMS) as a means of collecting gas stream particulates. The assessment included both experiments and analyses of theoretical models. Phase I included evaluations of theoretical ex...

  12. HIGH-GRADIENT MAGNETIC SEPARATION FOR REMOVAL OF SULFUR FROM COAL

    EPA Science Inventory

    The report gives results of a thorough physical, chemical, and magnetic characterization of a Pennsylvania coal from the Upper Freeport seam. The powdered coal was then subjected to high-gradient magnetic separations, as a function of magnetic field and fluid velocity, in both a ...

  13. 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.

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

    SciTech Connect

    Kawamura, K.; Okuwaki, A.; Verheyen, T.; Perry, G.J.

    2006-02-15

    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. 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.

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

    SciTech Connect

    Peter W. Carr; K.M. Fuller; D.R. Stoll; L.D. Steinkraus; M.S. Pasha; Glenn G. Hardin

    2005-12-30

    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 each 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.

  16. Coal liquefaction process streams characterization and evaluation: High performance liquid chromatography (HPLC) of coal liquefaction process streams using normal-phase separation with uv diode array detection

    SciTech Connect

    Clifford, D.J.; McKinney, D.E.; Hou, Lei; Hatcher, P.G.

    1994-01-01

    This study demonstrated the considerable potential of using two-dimensional, high performance liquid chromatography (HPLC) with normal-phase separation and ultraviolet (UV) diode array detection for the examination of filtered process liquids and the 850{degrees}F{sup {minus}} distillate materials derived from direct coal liquefaction process streams. A commercially available HPLC column (Hypersil Green PAH-2) provided excellent separation of the complex mixture of polynuclear aromatic hydrocarbons (PAHs) found in coal-derived process streams process. Some characteristics of the samples delineated by separation could be attributed to processing parameters. Mass recovery of the process derived samples was low (5--50 wt %). Penn State believes, however, that, improved recovery can be achieved. High resolution mass spectrometry and gas chromatography/mass spectrometry (GC/MS) also were used in this study to characterize the samples and the HPLC fractions. The GC/MS technique was used to preliminarily examine the GC-elutable portion of the samples. The GC/MS data were compared with the data from the HPLC technique. The use of an ultraviolet detector in the HPLC work precludes detecting the aliphatic portion of the sample. The GC/MS allowed for identification and quantification of that portion of the samples. Further development of the 2-D HPLC analytical method as a process development tool appears justified based on the results of this project.

  17. Direct Numerical Simulation of an Adverse Pressure Gradient Turbulent Boundary Layer at the Verge of Separation

    NASA Astrophysics Data System (ADS)

    Kitsios, Vassili; Atkinson, Callum; Sillero, Juan; Guillem, Borrell; Gungor, Ayse; Jimenéz, Javier; Soria, Julio

    2014-11-01

    We investigate the structure of an adverse pressure gradient (APG) turbulent boundary layer (TBL) at the verge of separation. The intended flow is generated via direct numerical simulation (DNS). The adopted DNS code was previously developed for a zero pressure gradient TBL. Here the farfield boundary condition (BC) is modified to generate the desired APG flow. The input parameters required for the APG BC are initially estimated from a series of Reynolds Averaged Navier-Stokes simulations. The BC is implemented into the DNS code with further refinement of the BC performed. The behaviour of the large scale dynamics is illustrated via the extraction of coherent structures from the DNS using analysis of the velocity gradient tensor and vortex clustering techniques. The authors acknowledge the research funding from the Australian Research Council and European Research Council, and the computational resources provided by NCI and PRACE.

  18. H.p.l.c. separation and study of the charge isomers of human placental glutathione transferase.

    PubMed Central

    Radulovic, L L; Kulkarni, A P

    1986-01-01

    Glutathione transferase (GST) from human placenta was purified by affinity chromatography and anion-exchange h.p.l.c. The enzyme exhibited different chromatographic and electrophoretic behaviours according to the concentration of GSH, suggesting a possible change in the net charge of the molecule and a concomitant conformational change due to ligand binding. Two interconvertible forms were quantitatively separated into distinct catalytically active states by h.p.l.c. Depending upon the GSH concentration, polyacrylamide-gel electrophoresis revealed the presence of one or two bands. A Kd of 0.42 mM for GSH was determined fluorimetrically. The loss in intrinsic fluorescence also suggested a conformational change in the enzyme. Kinetic studies using ethacrynic acid were conducted to determine whether the presumed conformational change could effect the catalytic capability of placental GST. A biphasic response in initial velocities was observed with increasing concentrations of GSH. Two apparent Km values of 0.38 and 50.27 mM were obtained for GSH, whereas Vmax. values showed a 46-fold difference. It was concluded that the enzyme assumes a highly anionic form in the presence of a low GSH concentration, whereas it is converted into relatively weaker anionic form when its immediate environment contains a high GSH concentration. Since the average tissue concentration of total GSH was estimated at 0.11 mM for term placenta, the results suggest that the high-affinity-low-activity conformer would predominate in vivo. Images Fig. 2. PMID:3800986

  19. 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. PMID:16318207

  20. Development and validation of an HPLC/UV assay for separation and quantification of peptide antigens from a liposomal vaccine delivery platform.

    PubMed

    Penwell, Andrea; Sharp, Kendall; Mansour, Marc; Sammatur, Leeladhar

    2012-07-01

    The development and validation of an HPLC method for the quantification of eight peptide antigens from the therapeutic cancer vaccine DPX-0907 is described. The antigens were formulated in DepoVax™, a patented liposomal vaccine delivery platform used in a phase 1 study for breast, ovarian, and prostate cancers. A gradient reversed-phase method with UV detection was optimized for separating and quantifying the peptide mixture. Several extraction methods investigated to extract the peptides from the lipids led to poor recovery of one or more of the peptides. A simple, reproducible, and high-recovery extraction procedure for the simultaneous quantification of hydrophilic and hydrophobic peptides was discovered using a liquid-liquid extraction with water-saturated n-butanol and sodium bicarbonate (0.1 M). The method was found to be specific, linear, accurate, precise, and reliable within the range of 50-150% of the nominal concentration for DPX-0907. The validated method was successfully applied to the assay of peptide content in pre-clinical and clinical batches of DPX-0907. PMID:22516680

  1. Evaluation of turbulence models for prediction of separated turbulent boundary layer under unsteady adverse pressure gradients

    NASA Astrophysics Data System (ADS)

    Park, Junshin; You, Donghyun

    2014-11-01

    Predicitive capabilites of Reynolds-averaged Navier-Stokes (RANS) techniques for separated flow under unsteady adverse pressure gradients have been assessed using SST k - ω model and Spalart-Allmaras model by comparing their results with direct numerical simulation (DNS) results. Both DNS and RANS have been conducted with a zero pressure gradient, a steady adverse pressure gradient, and an unsteady adverse pressure gradient, respectively. Comparative studies show that both RANS models predict earlier separation and fuller velocity profiles at the reattachment zone than DNS in the unsteady case, while reasonable agreements with DNS are observed for steady counterparts. Causes for differences in the predictive capability of RANS for steady and unsteady cases, are explained by examining the Reynolds stress term and eddy viscosity term in detail. The Reynolds stress and eddy viscosity are under-predicted by both RANS models in the unsteady case. The origin of the under-prediction of the Reynolds stress with both RANS models is revealed by investigating Reynolds stress budget terms obtained from DNS. Supported by the National Research Foundation of Korea Grant NRF-2012R1A1A2003699 and the Brain Korea 21+ program.

  2. Particle capture efficiency in a multi-wire model for high gradient magnetic separation

    NASA Astrophysics Data System (ADS)

    Eisenträger, Almut; Vella, Dominic; Griffiths, Ian M.

    2014-07-01

    High gradient magnetic separation (HGMS) is an efficient way to remove magnetic and paramagnetic particles, such as heavy metals, from waste water. As the suspension flows through a magnetized filter mesh, high magnetic gradients around the wires attract and capture the particles removing them from the fluid. We model such a system by considering the motion of a paramagnetic tracer particle through a periodic array of magnetized cylinders. We show that there is a critical Mason number (ratio of viscous to magnetic forces) below which the particle is captured irrespective of its initial position in the array. Above this threshold, particle capture is only partially successful and depends on the particle's entry position. We determine the relationship between the critical Mason number and the system geometry using numerical and asymptotic calculations. If a capture efficiency below 100% is sufficient, our results demonstrate how operating the HGMS system above the critical Mason number but with multiple separation cycles may increase efficiency.

  3. Two-dimensional electrophoresis of plant proteins with phastsystem using nonequilibrium pH gradient separation.

    PubMed

    Ferullo, J M; Nespoulous, L

    1991-10-01

    We have adapted a two-dimensional electrophoretic technique described by P. Z. O'Farrell et al. (Cell 12, 1133-1142, 1977) to Phastsystem, resolving both acidic and basic proteins by using nonequilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. Protein separation was optimized for the analysis of plant proteins. The use of the Phastsystem apparatus reduced times of preparation and separation, allowing the rapid screening of plant proteins on a large scale of isoelectric points. This technique was used for the immunodetection and characterization of two stress-induced proteins in irradiated tomato leaves. PMID:1789413

  4. Cr-polluted soil studied by high gradient magnetic separation and electron probe

    SciTech Connect

    Rikers, R.A.; Voncken, J.H.L.; Dalmijn, W.L.

    1998-12-01

    An Fe-rich soil from the site of a former leather tannery, heavily polluted with Cr, was studied using a combination of wet chemical analysis, high gradient magnetic separation (HGMS), and electron probe microanalysis (EPMA). It is demonstrated that such a combination is a powerful tool for the characterization of polluted soils, especially in cases where the pollution is present as discrete particles. Both EPMA and magnetic separation data indicated that the Cr pollution was present as a hydrous Cr-oxide phase. The Cr does not correlate with the Fe minerals, most likely as a result of the initial high Cr concentrations in the soil, which lead to precipitation of separate hydrous Cr-oxide minerals and Fe minerals. The Cr-containing material is present as (layered) aggregates, which are formed around larger quartz grains or around very small other particles that served as precipitation nuclei. Magnetic separation tests show that the Cr pollution can largely be removed by HGMS.

  5. Thermal Separation: Interplay between the Soret Effect and Entropic Force Gradient

    NASA Astrophysics Data System (ADS)

    Maeda, Yusuke T.; Buguin, Axel; Libchaber, Albert

    2011-07-01

    Thermophoresis, the Soret effect, depletes a high concentration of a polyethylene glycol polymer solution from the hot region and builds a concentration gradient. In such a solution, solutes of small concentration experience thermophoresis and polyethylene glycol concentration-dependent restoring forces. We report that by using focused laser heating and varying the polyethylene glycol concentration one observes geometrical localizations of solutes like DNA and RNA into patterns such as a ring. For DNA up to 5.6 kbp, the ring size decreases following a behavior analogous to a gel electrophoresis separation. Above 5.6 kbp, the ring diameter increases with the DNA length. Mixtures of DNA and RNA can be separated as well as different RNA lengths. Separation of colloids is also observed. The experiments might be relevant for the separation of small RNA ribozymes in an early stage of life.

  6. Optoelectrofluidic field separation based on light-intensity gradients and its applications

    NASA Astrophysics Data System (ADS)

    Yoon, Jinsung; Lee, Sanghyun; Kang, Kwan Hyoung

    2010-11-01

    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. 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. In addition, expanding the OEFS to biological applications such as rapid cell manipulation and separation will be discussed. The OEFS with LIG strategy could be a promising manipulation method of particles including biological cells in many applications where a rapid manipulation of particles over the entire working area is of interest.

  7. High gradient magnetic separation versus expanded bed adsorption: a first principle comparison.

    PubMed

    Hubbuch, J J; Matthiesen, D B; Hobley, T J; Thomas, O R

    2001-01-01

    A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the 'loaded' adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h(-1). Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h(-1) resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h(-1) raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption. PMID:11787803

  8. 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. PMID:11990566

  9. Facile synthesis of gradient mesoporous carbon monolith based on polymerization-induced phase separation

    NASA Astrophysics Data System (ADS)

    Xu, Shunjian; Luo, Yufeng; Zhong, Wei; Xiao, Zonghu; Luo, Yongping; Ou, Hui; Zhao, Xing-Zhong

    2014-06-01

    In this paper, a gradient mesoporous carbon (GMC) monolith derived from the mixtures of phenolic resin (PF) and ethylene glycol (EG) was prepared by a facile route based on polymerization-induced phase separation under temperature gradient (TG). A graded biphasic structure of PF-rich and EG-rich phases was first formed in preform under a TG, and then the preform was pyrolyzed to obtain the GMC monolith. The TG is mainly induced by the thermal resistance of the preferential phase separation layer at high temperature region. The pore structure of the monolith changes gradually along the TG direction. When the TG varies from 58°C to 29°C, the pore size, apparent porosity and specific surface area of the monolith range respectively from 18 nm to 83 nm, from 32% to 39% and from 140.5 m2/g to 515.3 m2/g. The gradient porous structure of the monolith is inherited from that of the preform, which depends on phase separation under TG in the resin mixtures. The pyrolysis mainly brings about the contraction of the pore size and wall thickness as well as the transformation of polymerized PF into glassy carbon.

  10. Separation of transition and heavy metals using stationary phase gradients and thin layer chromatography.

    PubMed

    Stegall, Stacy L; Ashraf, Kayesh M; Moye, Julie R; Higgins, Daniel A; Collinson, Maryanne M

    2016-05-13

    Stationary phase gradients for chelation thin layer chromatography (TLC) have been investigated as a tool to separate a mixture of metal ions. The gradient stationary phases were prepared using controlled rate infusion (CRI) from precursors containing mono-, bi-, and tri-dentate ligands, specifically 3-aminopropyltriethoxysilane, N-[3-(trimethoxysilyl)propyl] ethylenediamine, and N-[3-(trimethoxysilyl)propyl] diethylenetriamine. The presence and the extent of gradient formation were confirmed using N1s X-ray photoelectron spectroscopy (XPS). XPS results showed that the degree of modification was dependent on the aminosilane precursor, its concentration, and the rate of infusion. The separation of four transition and heavy metals (Co(2+), Pb(2+), Cu(2+), and Fe(3+)) on gradient and uniformly modified plates was compared using a mobile phase containing a stronger chelating agent, ethylenediaminetetraacetic acid (EDTA). The retention of the metal ions was manipulated by varying the surface concentration of the chelating ligands. The order of retention on unmodified plates and on plates modified with a monodentate ligand was Fe(3+)>Cu(2+)∼Pb(2+)∼Co(2+), while the order of retention on plates modified with bi- and tri-dentate ligands was Fe(3+)>Cu(2+)>Pb(2+)∼Co(2+). Fe(3+) and Cu(2+) were much more sensitive to the concentration of chelating ligand on the surface (displaying lower Rf values with increasing ligand concentration) than Pb(2+) and Co(2+). Complete separation was achieved using a high concentration of the tridentate ligand coupled with a longer time for modification, yielding a retention order of Fe(3+)>Cu(2+)>Co(2+)>Pb(2+). PMID:27090390

  11. The combination of analytical-scale HPLC separation with a TR-FRET assay to investigate JAK2 inhibitory compounds in a Boysenberry drink.

    PubMed

    McGhie, Tony K; Martin, Harry; Lunken, Rona C M

    2012-11-01

    We report the detection of JAK2 inhibitory activity in a Boysenberry (Rubus loganbaccus x R. baileyanus Britt.) drink using a combination of analytical-scale high performance liquid chromatography (HPLC) with a high-sensitivity time-resolved fluorescence coupled with fluorescence resonance energy transfer (TR-FRET) method. Phytochemical components of a Boysenberry drink were separated by reversed phase HPLC , and 84 separate fractions were collected. HPLC fractions corresponding to the ellagitannin and ellagic acid peaks observed in the chromatogram inhibited JAK2 activity. Anthocyanins, while they were the major phytochemical components of the Boysenberry drink, had no JAK2 inhibitory activity even though anthocyanins have previously been shown to be anti-inflammatory. This study demonstrates the usefulness of combining rapid analytical-scale HPLC separation with a highly sensitive fluorescence bioassay for characterising bioactivity in complex plant extracts. Ellagic acid was found to have an IC(50) of 92 nM against JAK2 and complete inhibition of JAK2 activity was observed in HPLC fractions of Boysenberry extract which had been diluted several hundred fold. To the best of our knowledge, this is the first demonstration that ellagitannins and other natural ellagic acid analogues are potent inhibitors of JAK2. Thus a drink containing Boysenberry juice concentrate may have anti-inflammatory properties. PMID:22899007

  12. 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. PMID:25869801

  13. 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

  14. 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. PMID:26890496

  15. An HPLC-MS/MS method for the separation of α-retinyl esters from retinyl esters.

    PubMed

    Goetz, Hilary J; Kopec, Rachel E; Riedl, Ken M; Cooperstone, Jessica L; Narayanasamy, Sureshbabu; Curley, Robert W; Schwartz, Steven J

    2016-09-01

    Enzymatic cleavage of the nonsymmetric provitamin A carotenoid α-carotene results in one molecule of retinal (vitamin A), and one molecule of α-retinal, a biologically inactive analog of true vitamin A. Due to structural similarities, α-retinyl esters and vitamin A esters typically coelute, resulting in the overestimation of vitamin A originating from α-carotene. Herein, we present a set of tools to identify and separate α-retinol products from vitamin A. α-Retinyl palmitate (αRP) standard was synthesized from α-ionone following a Wittig-Horner approach. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method employing a C30 column was then developed to separate the species. Authentic standards of retinyl esters and the synthesized α-RP confirmed respective identities, while other α-retinyl esters (i.e. myristate, linoleate, oleate, and stearate) were evidenced by their pseudomolecular ions observed in electrospray ionization (ESI) mode, fragmentation, and elution order. For quantitation, an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode was used, and retinol, the predominant in-source parent ion was selected and fragmented. The application of this method to a chylomicron-rich fraction of human plasma is demonstrated. This method can be used to better determine the quantity of vitamin A derived from foods containing α-carotene. PMID:27423669

  16. Optimization of IC Separation Based on Isocratic-to-Gradient Retention Modeling in Combination with Sequential Searching or Evolutionary Algorithm

    PubMed Central

    Rogošić, Marko; Šimović, Ena; Tišler, Vesna; Bolanča, Tomislav

    2013-01-01

    Gradient ion chromatography was used for the separation of eight sugars: arabitol, cellobiose, fructose, fucose, lactulose, melibiose, N-acetyl-D-glucosamine, and raffinose. The separation method was optimized using a combination of simplex or genetic algorithm with the isocratic-to-gradient retention modeling. Both the simplex and genetic algorithms provided well separated chromatograms in a similar analysis time. However, the simplex methodology showed severe drawbacks when dealing with local minima. Thus the genetic algorithm methodology proved as a method of choice for gradient optimization in this case. All the calculated/predicted chromatograms were compared with the real sample data, showing more than a satisfactory agreement. PMID:24349824

  17. Analysis of vortex flow in a cyclone separators based on the energy gradient theory

    NASA Astrophysics Data System (ADS)

    Liu, L.; Dou, H. S.; Chen, X. P.; Zhang, H. Z.

    2015-01-01

    Numerical simulation is carried out for the tangential inlet cyclone separator with different dimensionless parameter (the ratio of cone-tip diameter and cylinder diameter d/D is 0.342, 0.355, 0.368, 0.382 and 0.395). Then, the stability of the vortex flow in the separator is analyzed with the energy gradient theory. The governing equation is the Reynolds-averaged Navier-Stokes equations with Reynolds Stress Model (RSM). The finite volume method is employed to simulate the flow and the velocity, pressure, separation efficiency and the turbulent intensity are obtained. Results show that the location of the maximum tangential velocity is near the axis, and the highest outer vortex tangential velocity is achieved at the dimensionless rate of 0.355. When the d/D=0.355, the tangential velocity distribution in the outer vortex is the most close to velocity distribution characteristics of free vortex. The free vortex distribution has the best stability from the energy gradient theory which may be the reason why optimal performance can be achieved at d/D=0.355. To achieve high efficiency and low pressure drop for the device studied, d/D=0.355 is the optimal dimensionless diameter ratio.

  18. Separation of Ascites Myeloma Cells, Lymphocytes and Macrophages by Zonal Centrifugation on an Isokinetic Gradient

    PubMed Central

    Stewart, M. J.; Pretlow, T. G.; Hiramoto, Raymond

    1972-01-01

    In attempting to quantitate immunoglobulin synthesis by ascites myeloma cells, we were surprised to note that malignant appearing cells never exceeded 35.9 = 28.0% of ascitic cells and exceeded 22.4 ± 23.8% of ascitic cells on only one day between the transplantation of the tumor and the death of the host. The ascites tumor suspensions were separated primarily according to diameter, using a previously described isokinetic density gradient of Ficoll in tissue culture medium. This separation resulted in four modal populations of cells: red blood cells, lymphoid cells, macrophages and myeloma cells. The modal populations of macrophages and lymphoid cells always contained less than 0.2% myeloma cells. The purified cells were tested for tumorigenicity. The animal which received the largest number of cells from the macrophage zone received 296 times the number of cells which had been determined to be tumorigenic for myeloma cells. The animal which received the largest number of cells from the lymphoid zone received 1600 times the tumorigenic dose for myeloma cells. Neither of these animals has become ill 4 months after receiving the purified cells. We conclude that: a) Experimentalists who use ascites tumors are not justified in assuming that even easily detected quantitative differences between benign and malignant tissues would be reflected in analyses performed using unstandardized unexamined ascites tumor suspensions. b) In the case of the MOPC 104 mouse myeloma, cytologic criteria are adequate for distinguishing malignant cells from the inflammatory cells in the ascites suspension with a high degree of correspondence between cytologic appearance and biologic activity. c) Programmed gradient sedimentation in an isokinetic gradient of Ficoll in tissue culture medium is an effective means of separating malignant cells from benign cells in this particular ascites tumor. ImagesFig 1Fig 2Fig 3Fig 4 PMID:5080697

  19. HPLC method development for the online-coupling of chromatographic Perilla frutescens extract separation with xanthine oxidase enzymatic assay.

    PubMed

    Kaufmann, Christine M; Grassmann, Johanna; Letzel, Thomas

    2016-05-30

    Enzyme-regulatory effects of compounds contained in complex mixtures can be unveiled by coupling a continuous-flow enzyme assay to a chromatographic separation. A temperature-elevated separation was developed and the performance was tested using Perilla frutescens plant extracts of various polarity (water, methanol, ethanol/water). Owning to the need of maintaining sufficient enzymatic activity, only low organic solvent concentrations can be added to the mobile phase. Hence, to broaden the spectrum of eluting compounds, two different organic solvents and various contents were tested. The chromatographic performance and elution was further improved by the application of a moderate temperature gradient to the column. By taking the effect of eluent composition as well as calculated logD values and molecular structure of known extract compounds into account, unknown features were tentatively assigned. The method used allowed the successful observation of an enzymatic inhibition caused by P. frutescens extract. PMID:26986639

  20. DNS of self-similar adverse pressure gradient turbulent boundary layer at incipient separation

    NASA Astrophysics Data System (ADS)

    Soria, Julio; Kitsios, Vassili; Atkinson, Callum; Sillero, Juan; Borrell, Guillem; Gungar, Ayse; Jimenez, Javier

    2015-11-01

    A direct numerical simulation of a self-similar adverse pressure gradient turbulent boundary layer (APG-TBL) flow at incipient separation has been carried out. The maximum Reynolds number based on the momentum thickness, Reδ2 , reached in this DNS is 6,500. A wall-normal far-field boundary condition to effect the desired APG that will lead to the desired self-similar flow at the verge of separation has been developed. The self-similar analysis of the mean turbulent boundary layer equations yields the necessary conditions for a self-similar mean flow to exists. These conditions are tested using the DNS APG-TBL data base. First and second order statistics of the velocity across the APG-TBL are also presented in the light of the self-similar analysis results and compared to the results of a zero pressure gradient turbulent boundary layer DNS with similar mean inflow characteristics as the APG-TBL. The support of the ARC, NCI and Pawsey SCC funded by the Australian and Western Australian governments as well as the support of PRACE funded by the European Union are gratefully acknowledged.

  1. 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. PMID:25828094

  2. Protein separation and characterization by np-RP-HPLC followed by intact MALDI-TOF mass spectrometry and peptide mass mapping analyses

    PubMed Central

    Dauly, Claire; Perlman, David H.; Costello, Catherine E.; McComb, Mark E.

    2008-01-01

    Due to their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of non-porous-reversed-phase (np-RP) HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF MS was in the 10–50 pg level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall we demonstrate that np-RP HPLC followed by MALDI-TOF MS allows for rapid, sensitive and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information. PMID:16823977

  3. Improved high gradient magnetic separation for the positive selection of human blood mononuclear cells using ordered wire filters

    NASA Astrophysics Data System (ADS)

    Richards, A. J.; Thomas, T. E.; Roath, O. S.; Watson, J. H. C.; Smith, R. J. S.; Lansdorp, P. M.

    1993-04-01

    CD8 + lymphocytes, fresh and previously cryopreserved, from ficolled human peripheral blood have been enriched to 99% purity with 68% recovery. The CD8 + lymphocytes were labelled with colloidal, magnetisable, Dextran-Iron particles using Tetrameric Antibody complexes. The labelled cells were magnetically separated using High Gradient Magnetic Separation with novel, ordered filters.

  4. Performance of popular turbulence models for attached and separated adverse pressure gradient flows

    NASA Technical Reports Server (NTRS)

    Menter, F. R.

    1991-01-01

    The performance of four popular eddy-viscosity turbulence models under adverse pressure gradient conditions is investigated. The Baldwin-Lomax, the Johnson-King, the Baldwin-Barth, and the Wilcox-omega models have been implemented into the INS code, which solves the incompressible Reynolds-averaged Navier-Stokes equations. Results are shown for the well known Samuel-Joubert flow and two new flowfields, recently reported by D. M. Driver (1991). The two new flowfields pose a stronger test of the models than the Samuel-Joubert flow, because of the more severe retardation of the boundary layer, including separation in one case. A detailed comparison of the numerical results and the experimental data is shown.

  5. Performance of popular turbulence models for attached and separated adverse pressure gradient flows

    NASA Astrophysics Data System (ADS)

    Menter, F. R.

    1991-06-01

    The performance of four popular eddy-viscosity turbulence models under adverse pressure gradient conditions is investigated. The Baldwin-Lomax, the Johnson-King, the Baldwin-Barth, and the Wilcox-omega models have been implemented into the INS code, which solves the incompressible Reynolds-averaged Navier-Stokes equations. Results are shown for the well known Samuel-Joubert flow and two new flowfields, recently reported by D. M. Driver (1991). The two new flowfields pose a stronger test of the models than the Samuel-Joubert flow, because of the more severe retardation of the boundary layer, including separation in one case. A detailed comparison of the numerical results and the experimental data is shown.

  6. 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. PMID:27092965

  7. Determination of arsenic species in edible periwinkles (Littorina littorea) by HPLC-ICPMS and XAS along a contamination gradient

    SciTech Connect

    Whaley-Martin, K. J.; Koch, I.; Reimer, K. J.

    2013-06-12

    Arsenic is naturally found in the tissues of marine animals, usually as the non-toxic arsenical arsenobetaine, but exposure to elevated arsenic concentrations in the environment may alter the arsenic species distribution within tissues of the organism. This study examined the arsenic species in the tissues of the marine periwinkle (Littorina littorea) along an arsenic concentration gradient in the sediment. The arsenicals in L. littorea were examined using the complementary analytical methods high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC–ICPMS) and X-ray absorption spectroscopy (XAS). Total arsenic concentrations in the periwinkle tissues ranged from 56 to 840 mg · kg-1 dry weight (equivalent to 13 to 190 mg · kg-1 wet weight). Inorganic arsenicals were found to be positively correlated with total arsenic concentrations (R2 = 0.993) and reached 600 mg · kg-1 dry weight, the highest reported to date in marine organisms. These high inorganic arsenic concentrations within this low trophic organism pose a potential toxicological risk to higher trophic consumers.

  8. HPLC Determination of Taurine in Sports Drinks

    NASA Astrophysics Data System (ADS)

    Orth, Dale L.

    2001-06-01

    The amino acid taurine (2-aminoethanesulfonic acid) is present as a nutritional supplement in many sports drinks. An experiment, suitable for a junior-senior level instrumental analysis course, is described to measure the amount of taurine in these sports drinks. A pre-column derivatization with Sanger's reagent, 2,4-dinitrofluorobenzene, is followed by an HPLC separation utilizing a gradient elution, and detection at 360 nm.

  9. Functional characterization of human thymocyte subpopulations separated by density gradient centrifugation.

    PubMed Central

    Lederman, H M; Lee, J W; Cohen, A; Gelfand, E W

    1984-01-01

    A bovine serum albumin gradient was used to separate two populations of human thymocytes--a minority population (8%) of large thymocytes (LT) and a majority population (92%) of small thymocytes (ST). Fifty per cent of LT cells were in the S, G2 or M phases of the cell cycle compared to 5% of ST cells and 15% of unfractionated thymocytes. LT cells proliferated in response to T cell mitogens and included all of the T colony precursor cells (TCPC). In contrast, ST cells proliferated with mitogens only in the presence of added T cell growth factors and contained none of the thymocyte TCPC. ST cells neither helped nor suppressed the function of LT cells in any assay. This separation technique has provided a rapid method for isolating functionally distinct thymic lymphocyte subpopulations and permitted a further definition of the TCPC in the human thymus. Furthermore it should prove useful in studies of thymocytes at different stages of the cell cycle. PMID:6607793

  10. A new turbulence closure model for boundary layer flows with strong adverse pressure gradients and separation

    NASA Technical Reports Server (NTRS)

    Johnson, D. A.; King, L. S.

    1984-01-01

    A new turbulence closure model designed specifically to treat two-dimensional, turbulent boundary layers with strong adverse pressure gradients and attendant separation, is presented. The influence of history effects are modeled by using an ordinary differential equation (ODE) derived from the turbulence kinetic-energy equation, to describe the streamwise development of the maximum Reynolds shear stress in conjunction with an assumed eddy-viscosity distribution which has as its velocity scale the maximum Reynolds shear stress. In the outer part of the boundary layer, the eddy viscosity is treated as a free parameter which is adjusted in order to satisfy the ODE for the maximum shear stress. Because of this, the model s not simply an eddy-viscosity model, but contains features of a Reynolds-stress model. Comparisons with experiments are presented which clearly show the proposed model to be superior to the Cebeci-Smith model in treating strongly retarded and separated flows. In contrast to two-equation, eddy-viscosity models, it requires only slightly more computational effort than simple models like the Cebeci-Smith model.

  11. 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. PMID:26907700

  12. Volume based vs. time based chromatograms: reproducibility of data for gradient separations under high and low pressure conditions.

    PubMed

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

    2014-05-23

    A critical aspect in fast gradient separations carried out under constant pressure, in the very high pressure liquid chromatography (VHPLC) mode is that time-based chromatograms may not yield highly reproducible separations. A proposed solution to improve the reproducibility of these separations involves plotting the chromatograms as functions of the volume eluted vs. UV absorbance instead of time vs. UV. To study the consequences of using the volume-based rather than the time-based chromatograms, separations were first performed under low pressures that do not generate significant amounts of heat and for which the variations of the eluent density along the columns are negligible. Secondly, they were performed under very high pressures that do generate heat and measurable variations of the local retention factor and eluent density along the column. Comparison of the results provides estimates of the improvements obtained when volume based chromatograms are used in gradient analyses. Using a column packed with fully porous particles, four different types of methods and several sets for each method were used to perform the gradient elution runs: two sets of constant flow rate operations, four sets of constant pressure operations, two sets of constant pressure operations with programmed flow rate, and one set using the constant heat loss approach. The differences between time-based and volume-based chromatograms are demonstrated by using eight replicates of early, middle, and last eluting peaks. The results show that volume-based chromatograms improve the retention time reproducibility of the four constant pressure methods by a factor of 3.7 on average. If the column is not thermally conditioned prior to performing a long series of separations, flow controlled methods (constant flow rate, programmed constant pressure, and constant wall heat approaches) are more precise. If one gradient run is used to bring the column to a relatively stable temperature, constant

  13. Conventional Chiralpak ID vs. capillary Chiralpak ID-3 amylose tris-(3-chlorophenylcarbamate)-based chiral stationary phase columns for the enantioselective HPLC separation of pharmaceutical racemates.

    PubMed

    Ahmed, Marwa; Gwairgi, Marina; Ghanem, Ashraf

    2014-11-01

    A comparative enantioselective analysis using immobilized amylose tris-(3-chlorophenylcarbamate) as chiral stationary phase in conventional high-performance liquid chromatography (HPLC) with Chiralpak ID (4.6 mm ID × 250 mm, 5 µm silica gel) and micro-HPLC with Chiralpak ID-3 (0.30 mm ID × 150 mm, 3 µm silica gel) was conducted. Pharmaceutical racemates of 12 pharmacological classes, namely, α- and β-blockers, anti-inflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminics, anticancer drugs, and antiarrhythmic drugs were screened under normal phase conditions. The effect of an organic modifier on the analyte retentions and enantiomer recognition was investigated. Baseline separation was achieved for 1-acenaphthenol, carprofen, celiprolol, cizolirtine carbinol, miconazole, tebuconazole, 4-hydroxy-3-methoxymandelic acid, 1-indanol, 1-(2-chlorophenyl)ethanol, 1-phenyl-2-propanol, flavanone, 6-hydroxyflavanone, 4-bromogluthethimide, and pentobarbital on the 4.6 mm ID packed with a 5 µm silica column using conventional HPLC. Nonetheless, baseline separation was achieved for aminoglutethimide, naftopidil, and thalidomide on the 0.3 mm ID packed with a 3 µm silica capillary column. PMID:25271972

  14. Evaluation of the potential of nonlinear gradients for separating a ternary mixture.

    PubMed

    Damtew, Andualem; Sreedhar, Balamurali; Seidel-Morgenstern, Andreas

    2009-07-10

    The potential of applying nonlinear gradients in preparative chromatography is evaluated theoretically and experimentally. The second eluting component of a ternary model mixture is considered as the target component. Systematic gradient resolution experiments were conducted for this mixture using reversed phase chromatography. In preliminary experimental investigations the effect of the mobile phase composition on the specific distribution equilibria was quantified. Concave and convex solvent strength gradients were analysed and compared with conventional linear gradients. Main result of the study is the fact that significant improvement in productivity can be achieved if the gradient shapes are designed carefully. PMID:19501828

  15. TREATMENT OF COMBINED SEWER OVERFLOWS BY HIGH GRADIENT MAGNETIC SEPARATION. ON-SITE TESTING WITH MOBILE PILOT PLANT TRAILER

    EPA Science Inventory

    Seeded water treatment using a SALA high gradient magnetic separator pilot plant system was conducted on combined sewer overflows and raw sewage at SALA Magnetics in Cambridge, MA and at on-site locations in the Boston area. Special emphasis was placed on specific design and oper...

  16. Effect of stationary phase structure on retention and selectivity tuning in the high-throughput separation of tocopherol isomers by HPLC.

    PubMed

    Buszewski, Boguslaw; Krupczynska, Katarzyna; Bazylak, Grzegorz

    2004-06-01

    Four stationary phases containing different groups such as: C18, C30, alkylamide, and cholesterolic, were presented for simultaneous HPLC analysis of structural isomers of tocopherol. Especially, the influence of stationary phase structure and properties on tuning of the highly selective HPLC separation of beta- and gamma-tocopherol pair demonstrating, respectively, para- and ortho- arrangement of methyl substituents on the 6-chromanol ring, has been elucidated. It was pointed out that selectivity of each stationary phase has been a result of modulation in the mass transfer and set of unspecific interactions in the tertiary system comprising analyte <==> stationary phase <==> mobile phase. Differences in observed retention and specific selectivity of tocopherols together with the stationary phase structure investigations indicated that a spatial organization changing of chemically bonded ligands as predominantly a solvation consequence. Additional molecular modeling studies preliminary explained some of these complicated supramolecular phenomena which caused that cholesterolic stationary phase offered beneficial performance in screening of tocopherols by HPLC and biomimetic studies of not completely recognized interactions of tocopherol isomers and biological membranes. PMID:15200386

  17. 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.

  18. 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.

  19. Application of high gradient magnetic separation principles to magnetic drug targeting

    NASA Astrophysics Data System (ADS)

    Ritter, James A.; Ebner, Armin D.; Daniel, Karen D.; Stewart, Krystle L.

    2004-09-01

    A hypothetical magnetic drug targeting system, utilizing high gradient magnetic separation (HGMS) principles, was studied theoretically using FEMLAB simulations. This new approach uses a ferromagnetic wire placed at a bifurcation point inside a blood vessel and an externally applied magnetic field, to magnetically guide magnetic drug carrier particles (MDCP) through the circulatory system and then to magnetically retain them at a target site. Wire collection (CE) and diversion (DE) efficiencies were defined and used to evaluate the system performance. CE and DE both increase as the strength of the applied magnetic field (0.3-2.0 T), the amount of ferromagnetic material (iron) in the MDCP (20-100%) and the size of the MDCP (1-10 μm radius) increase, and as the average inlet velocity (0.1-0.8 m s-1), the size of the wire (50-250 μm radius) and the ratio (4-10) of the parent vessel radius (0.25-1.25 mm radius) to wire radius decrease. The effect of the applied magnetic field direction (0° and 90°) on CE and DE was minimal. Under these plausible conditions, CEs as high as 70% were obtained, with DEs reaching only 30%; however, when the MDCPs were allowed to agglomerate (4-10 μm radius), CEs and DEs of 100% were indeed achieved. These results reveal that this new magnetic drug targeting approach for magnetically collecting MDCPs at a target site, even in arteries with very high velocities, is feasible and very promising; this new approach for magnetically guiding MDCPs through the circulatory system is also feasible but more limited. Overall, this study shows that magnetic drug targeting, based on HGMS principles, has considerable promise as an effective drug targeting tool with many potential applications.

  20. Improved separation and quantification of neutral and polar lipid classes by HPLC-ELSD using a monolithic silica phase: application to exceptional marine lipids.

    PubMed

    Graeve, Martin; Janssen, Dieter

    2009-07-01

    An improved HPLC method is presented, which allows separation and quantification of a broad range of lipid classes of marine zooplankton with special regard to neutral lipids. Marine zooplankton species often produce high amounts of exceptional lipids, especially at high latitudes, in order to cope with the harsh environmental conditions and strong seasonality in food supply. Major neutral lipid classes are wax esters, triacylglycerols, diacylglycerol ethers, free fatty alcohols and sterols. Neutral and polar lipids were separated and identified on a monolithic silica column (Chromolith Performance-Si) using high performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). The method resolves a broad spectrum of lipids, varying in polarity from squalene to lysophosphatidylcholine in a single run. The total run time was 35 min including column re-equilibration. The calibration was made at levels of 0.1-60 microg lipid/injection, but a 10-15-fold greater amount can be injected if single lipid classes need to be separated, e.g. for further determination of individual fatty acids. The method was applied to representative Arctic zooplankton species (copepods, pteropods, euphausiids and ctenophores) that are known to biosynthesize in particular neutral lipids like diacylglycerol ethers and free fatty alcohols. PMID:19493709

  1. Polymer networks with bicontinuous gradient morphologies resulting from the competition between phase separation and photopolymerization.

    PubMed

    Hirose, Atsuko; Shimada, Keisuke; Hayashi, Chie; Nakanishi, Hideyuki; Norisuye, Tomohisa; Tran-Cong-Miyata, Qui

    2016-02-14

    Poly(ethyl acrylate)/poly(methyl methacrylate) (PEA/PMMA) polymer networks (IPNs) with spatially graded bicontinuous morphology were designed and controlled by taking advantage of the spinodal decomposition process induced by photopolymerization of the MMA monomer. Spatial gradients of the quench depth, induced by the gradients of light intensity, were generated along the path of the excitation light travelling through the mixture. Bicontinuous structures with uniaxial gradient of characteristic length scales were obtained by two different methods: simply irradiating the mixture with strong light intensity along the Z-direction and using the so-called computer-assisted irradiation (CAI) method with moderate intensity to generate the light intensity gradient exclusively in the XY plane. These experimental results suggest that the combination of these two irradiation methods could provide polymer materials with biaxially co-continuous gradient morphology. An analysis method using the concept of spatial correlation function was developed to analyze the time-evolution of these graded structures. The experimental results obtained in this study suggest a promising method to design gradient polymers in the bulk state (3D) as well as on the surface (2D) by taking advantage of photopolymerization. PMID:26738621

  2. Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment.

    PubMed

    Grøndahl, Frøy; Tveit, Heidi; Akslen-Hoel, Linn Kristin; Prydz, Kristian

    2011-01-01

    The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports. PMID:21126737

  3. Comparison of displacement versus gradient mode for separation of a complex protein mixture by anion-exchange chromatography.

    PubMed

    Ahrends, Robert; Lichtner, Björn; Buck, Friedrich; Hildebrand, Diana; Kotasinska, Marta; Kohlbacher, Oliver; Kwiatkowski, Marcel; Wagner, Moritz; Trusch, Maria; Schlüter, Hartmut

    2012-07-15

    Liquid chromatography is often the method of choice for the analysis of proteins in their native state. Nevertheless compared to two-dimensional electrophoresis, the resolution of common chromatographic techniques is low. Liquid chromatography in the displacement mode has previously been shown to offer higher resolution and to elute proteins in the high concentrations. In this study we compared to what extend displacement mode was a suitable alternative to gradient mode for the separation of a complex protein mixture using anion-exchange displacement chromatography and if it is therefore helpful for proteomic investigations. Hence we analyzed the qualitative protein composition of each fraction by tryptic digestion of the proteins, analysis of the tryptic peptides by liquid chromatography coupled to mass spectrometry followed by data base analysis and by measuring the elution profiles of 22 selected proteins with selected reaction monitoring mass spectrometry. In the fractions of displacement mode a significantly higher number of identified proteins (51 versus 16) was yielded in comparison to gradient mode. The resolution of displacement chromatography was slightly lower than of gradient chromatography for many but not for all proteins. The selectivities of displacement mode and gradient mode are very different. In conclusion displacement chromatography is a well suited alternative for top-down proteomic approaches which start with separating intact proteins first prior to mass spectrometric analysis of intact or digested proteins. The significant orthogonality of both modes may be used in the future for combining them in multidimensional fractionation procedures. PMID:22727752

  4. Removal and recycle of phosphate from treated water of sewage plants with zirconium ferrite adsorbent by high gradient magnetic separation

    NASA Astrophysics Data System (ADS)

    Ito, D.; Nishimura, K.; Miura, O.

    2009-03-01

    Zirconium ferrite particles are good adsorbent for phosphate ions. Magnetic separation characteristics for removal of phosphate from treated water of sewage plants with the adsorbent have been studied to prevent eutrophication of semi-enclosed bay, e.g. the bay of Tokyo. Based on the adsorption for the phosphate ions and ferromagnetic properties of the zirconium ferrite adsorbent, high gradient magnetic separation characteristics with using superconducting magnet was discussed. Very rapid magnetic filtration velocity, i.e. 1m/s, and regeneration properties of the adsorbent indicate that the zirconium ferrite is the excellent adsorbent for phosphorus removal and recycle from treated water of large scale sewage plants.

  5. Simultaneous Separation of Negatively and Positively Charged Species in Dynamic Field Gradient Focusing Using a Dual Polarity Electric Field

    PubMed Central

    Burke, Jeffrey M.; Huang, Zheng; Ivory, Cornelius F.

    2011-01-01

    Dynamic field gradient focusing (DFGF) utilizes an electric field gradient established by a computer-controlled electrode array to separate and concentrate charged analytes at unique axial positions. Traditionally, DFGF has been restricted to the analysis of negatively charged species due to limitations in the software of our voltage controller. This paper introduces a new voltage controller capable of operating under normal polarity (positive potentials applied to the electrode array) and reversed polarity (negative potentials applied to the electrode array) for the separation of negatively and positively charged analytes, respectively. The experiments conducted under normal polarity and reversed polarity illustrate the utility of the new controller to perform reproducible DFGF separations (elution times showing less than 1% run-to-run variation) over a wide pH range (3.08 to 8.5) regardless of the protein charge. A dual polarity experiment is then shown in which the separation channel has been divided into normal polarity and reversed polarity regions. This simultaneous separation of negatively charged R-phycoerythrin (R-PE) and positively charged cytochrome c (CYTC) within the same DFGF apparatus is shown. PMID:19722517

  6. SUPERCONDUCTING OPEN-GRADIENT MAGNETIC SEPARATION FOR THE PRETREATMENT OF RADIOACTIVE OR MIXED WASTE VITRIFICATION FEEDS

    EPA Science Inventory

    Scientists need to gain a better understanding of the magnetic separation processes that can be used to separate deleterious constituents (crystalline, amorphous, and colloidal) in vitrification feed streams for borosilicate glass production without adding chemicals or generating...

  7. Optimisation of the separation of herbicides by linear gradient high performance liquid chromatography utilising artificial neural networks.

    PubMed

    Tran, Anh T K; Hyne, Ross V; Pablo, Fleur; Day, W Roy; Doble, P

    2007-02-28

    An artificial neural network (ANN) was employed to model the chromatographic response surface for the linear gradient separation of 10 herbicides that are commonly detected in storm run-off water in agricultural catchments. The herbicides (dicamba, simazine, 2,4-D, MCPA, triclopyr, atrazine, diuron, clomazone, bensulfuron-methyl and metolachlor) were separated using reverse phase high performance liquid chromatography and detected with a photodiode array detector. The ANN was trained using the pH of the mobile phase and the slope of the acetonitrile/water gradient as input variables. A total of nine experiments were required to generate sufficient data to train the ANN to accurately describe the retention times of each of the herbicides within a defined experimental space of mobile phase pH range 3.0-4.8 and linear gradient slope 1-4% acetonitrile/min. The modelled chromatographic response surface was then used to determine the optimum separation within the experimental space. This approach allowed the rapid determination of experimental conditions for baseline resolution of all 10 herbicides. Illustrative examples of determination of these components in Milli-Q water, Sydney mains water and natural water samples spiked at 0.5-1mug/L are shown. Recoveries were over 70% for solid-phase extraction using Waters Oasis((R)) HLB 6cm(3) cartridges. PMID:19071444

  8. Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis.

    PubMed

    Vézina, C A; Milne, R W; Weech, P K; Marcel, Y L

    1988-05-01

    The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A

  9. 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. PMID:26405759

  10. 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

  11. Development of Novel RP-HPLC Method for Separation and Estimation of Critical Geometric Isomer and Other Related Impurities of Tafluprost Drug Substance and Identification of Major Degradation Compounds by Using LC-MS.

    PubMed

    Sreenivasulu, J; Venkata Ramana, P; Sampath Kumar Reddy, G; Rakesh, M; Nagaraju, Ch V S; Thirumalai Rajan, S; Eswaraiah, S; Kishore, M; Ramakrishna, M

    2016-09-01

    A novel, simple, sensitive and stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative determination of the geometric isomer (Trans) and other related substances in the active pharmaceutical ingredient (API) of Tafluprost (TFL), with their determination by an assay. A chromatographic separation of TFL and its impurities was achieved with a C18 analytical column, using gradient elution with mobile phase A consisting of a mixture of water, methanol and orthophosphoric acid (900:100:1, v/v) and mobile phase B consisting of a mixture of acetonitrile and water (900:100, v/v). The instrumental settings included a flow rate of 1.0 mL/min for related substances and 1.2 mL/min for the assay, a column temperature of 50°C and a detector wavelength of 210 nm, using a photodiode array detector. TFL was exposed to thermal, photolytic, hydrolytic and oxidative stress conditions and the stressed samples were analyzed by the proposed method. Peak homogeneity data of TFL were obtained by using a photodiode array detector in the stressed sample chromatograms, which demonstrated the specificity of the method for estimation in the presence of degradants. The developed method was validated for parameters such as precision, accuracy, linearity, limit of detection, limit of quantification, ruggedness and robustness as per ICH guidelines. PMID:27226462

  12. Comparison of ultracentrifugation and density gradient separation methods for isolating Tca8113 human tongue cancer cell line-derived exosomes

    PubMed Central

    ZHANG, ZHUOYUAN; WANG, CHENXING; LI, TANG; LIU, ZHE; LI, LONGJIANG

    2014-01-01

    The aim of the present study was to compare the method of ultracentrifugation and density gradient separation for isolating Tca8113 human tongue squamous cell carcinoma cell line-derived exosomes. The exosomes were obtained from the culture supernatant of cultured Tca8113 cells, respectively, followed by identification with transmission electron microscopy observation and western blot analysis. The two different methods were then compared by the morphology, the distribution range of the particle size and the concentration of proteins of the extracted exosomes. In vitro, Tca8113 cells can secrete a large amount of vesicle-like structures, which are identified as exosomes by the presence of the surface markers, Hsp-70 and Alix. The protein profile of the two products are almost the same, however the particle size distribution of the exosomes extracted with density gradient centrifugation are more limited, between 40–120 nm, and these have a higher protein concentration. The results indicate that Tca8113 cells can secrete exosomes in vitro, and the density gradient separation methods for purifying exosomes is improved, which is helpful for future research and application of exosomes. PMID:25202395

  13. 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.

  14. A Computational Study on the Effects of Dynamic Roughness Application to Separated Transitional Flows Affected by Adverse Pressure Gradient

    NASA Astrophysics Data System (ADS)

    Campitelli, Gennaro

    The study of transitional flows is considered crucial for many practical engineering applications. In fact, a comprehensive understanding of the laminar-turbulent transition phenomenon often helps to improve the overall performance of apparatuses such as airfoils, wind turbines, hulls and turbomachinery blades. In addition to understanding and prediction of transitional flows, active research continues in the area of boundary layer control, which includes control of phenomena such as flow separation and transition. For instance, optimum geometrical shaping may be followed by the adoption on the wall-surface of riblets to adjust pressure gradient and reduce drag. Further "flow control" may also be acquired by introducing active devices able to modify the flow field in order to accomplish a desired aerodynamic task. Such flow manipulation is often achieved by using time-dependent forcing mechanisms which promote natural instabilities amplifying the control effectiveness. Localized energy inputs such as Lorentz-force actuator, piezoelectric flaps and synthetic jets all produce a consistent boundary layer mixing enhancement with lift increase and drag abatement. The current numerical study attempts to demonstrate the efficacy of dynamic roughness (DR) on altering separated-reattached transitional flows under adverse pressure gradient. It has already been proven how DR, acting on the boundary sublayer perturbation, is able to suppress (partially or completely) the typical leading edge separation for an airfoil at different angles of attack. This makes DR particularly suitable for separated flow control applications where the shear layer reattaches presenting the characteristic laminar separation bubble. A numerical sensitivity study has been conducted with an efficient orthogonal design taking into account four different control parameters on three levels (actuation frequency, humps height, rows displacement, synchronization) to provide an optimum DR setup which limits

  15. 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.

  16. Development of a membrane-less dynamic field gradient focusing device for the separation of low-molecular-weight molecules

    PubMed Central

    Burke, Jeffrey M.; Smith, Colin D.; Ivory, Cornelius F.

    2010-01-01

    Dynamic field gradient focusing uses an electric field gradient generated by controlling the voltage profile of an electrode array to separate and concentrate charged analytes according to their individual electrophoretic mobilities. This study describes a new instrument in which the electrodes have been placed within the separation channel. The major challenge faced with this device is that when applied voltages to the electrodes are larger than the redox potential of water, electrolysis will occur, producing hydrogen ions (H+) plus oxygen gas on the anodes and hydroxide (OH−) plus hydrogen gas on the cathodes. The resulting gas bubbles and pH excursions can cause problems with system performance and reproducibility. An on-column, degassing system that can remove gas bubbles “on-the-fly” is described. In addition, the use of a high capacity, low-conductivity buffer to address the problem of the pH shift that occurs due to the production of H+ on the anodes is illustrated. Finally, the successful separation of three, low-molecular-weight dyes (amaranth, bromophenol blue and methyl red) is described. PMID:20191553

  17. Active control of Boundary Layer Separation & Flow Distortion in Adverse Pressure Gradient Flows via Supersonic Microjets

    NASA Technical Reports Server (NTRS)

    Alvi, Farrukh S.; Gorton, Susan (Technical Monitor)

    2005-01-01

    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 aim of this research has been to understand the nature of separation and more importantly, to explore techniques to actively control this flow separation. In particular, the use of supersonic microjets as a means of controlling boundary layer separation was explored. The geometry used for the early part of this study was a simple diverging Stratford ramp, equipped with arrays of supersonic microjets. Initial results, based on the mean surface pressure distribution, surface flow visualization and Planar Laser Scattering (PLS) indicated a reverse flow region. We implemented supersonic microjets to control this separation and flow visualization results appeared to suggest that microjets have a favorable effect, at least to a certain extent. However, the details of the separated flow field were difficult to determine based on surface pressure distribution, surface flow patterns and PLS alone. It was also difficult to clearly determine the exact influence of the supersonic microjets on this flow. In the latter part of this study, the properties of this flow-field and the effect of supersonic microjets on its behavior were investigated in further detail using 2-component (planar) Particle Image Velocimetry (PIV). The results clearly show that the activation of microjets eliminated flow separation and resulted in a significant increase in the momentum of the fluid near the ramp surface. Also notable is the fact that the gain in momentum due to the elimination of flow separation is at least an order of magnitude larger (two orders of magnitude larger in most cases) than the momentum injected by the microjets and is accomplished with very

  18. Separation and quantitative determination of cinacalcet metabolites in urine sample using RP-HPLC after derivation with a fluorescent labeling reagent.

    PubMed

    Farnoudian-Habibi, Amir; Jaymand, Mehdi

    2016-08-01

    In this investigation, a novel strategy for separation and quantitative determination of four metabolites of cinacalcet (M2a-Glu, M2b-Glu, M7-Gly, and M8-Gly) in human urine is suggested. The analytical assay is based on a pre-column derivation procedure of cinacalcet metabolites with 1-pyrenyldiazomethane (PDAM) as a fluorescent labeling reagent, and subsequently separation and quantitative determination with reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a fluorescence detector. Metabolites were separated on a Microsorb-MV 100-5 C18 chromatography column (250×4.6mm, 5μm) using acetate buffer (pH 3.5):methanol (30:70 v/v) as mobile phase at a flow rate of 1.0mLmin(-1). The method was fully validated in terms of linearity (r(2)>0.996; 1-10ngmL(-1)), precision (both intra-day and inter-day; RSD<6.2%), accuracy (92-110%), specificity, robustness (0.15%

  19. Separation and identification of cis and trans isomers of 2-butene-1,4-diol and lafutidine by HPLC and LC-MS*

    PubMed Central

    Pan, Chun-xiu; Xu, Xiu-zhu; He, Hong-mei; Cai, Xiao-jun; Zhang, Xue-jun

    2005-01-01

    The cis and trans isomers separation of 2-butene-1,4-diol and lafutidine were studied by HPLC on two kinds of chiral columns: (S,S)-Whelk-O 1 and ChiraSpher. The isomers of 2-butene-1,4-diol can be separated on both chiral columns while the isomers of lafutidine can only be resolved on ChiraSpher column. The influence of different type and amount of mobile phase modifier on the isomers separation was extensively studied. The resolution of cis and trans isomers of 2-butene-1,4-diol was 2.61 on (S,S)-Whelk-O 1 column with hexane-ethanol (97:3, v/v) as the mobile phase. The resolution of lafutidine was 1.89 on ChiraSpher column with hexane-ethanol-THF-diethylamine (92:3:5:0.1, v/v/v/v) as the mobile phase. LC-MS methods were developed to identify the isomer peaks. PMID:15593397

  20. Investigation of the process of diamagnetic particle separation in a high-gradient ordered-structure magnetic field

    NASA Astrophysics Data System (ADS)

    Kashevskii, B. É.; Kashevskii, S. É.; Prokhorov, I. V.; Zholud', A. M.

    2011-05-01

    On the basis of the model of a flow-type magnetic filter with a transversely magnetized ordered system of long ferromagnetic rods of rectangular cross section, the process of high-gradient magnetic separation of microscopic diamagnetic particles (potato starch granules of sizes 8-30 μm) from a liquid suspension has been investigated. The registered laws of change in the concentration and size distribution of particles at the suspension outlet from the filter agree with the theoretical conclusions obtained from the analysis of the magnetic field structure and thecharacter of the particle motion in the filter volume.

  1. Quantitative determination and pattern recognition analyses of bioactive marker compounds from Dipsaci Radix by HPLC.

    PubMed

    Zhao, Bing Tian; Jeong, Su Yang; Moon, Dong Cheul; Son, Kun Ho; Son, Jong Keun; Woo, Mi Hee

    2013-11-01

    In this study, quantitative and pattern recognition analyses were developed using HPLC/UV for the quality evaluation of Dipsaci Radix. For quantitative analysis, five major bioactive compounds were assessed. The separation conditions employed for HPLC/UV were optimized using ODS C18 column (250 × 4.6 mm, 5 μm) with a gradient of acetonitrile and water as the mobile phase at a flow rate of 1.0 mL/min and a detection wavelength of 212 nm. These methods were fully validated with respect to linearity, accuracy, precision, recovery, and robustness. The HPLC/UV method was applied successfully to the quantification of five major compounds in the extract of Dipsaci Radix. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of 17 Dipsaci Radix and four Phlomidis Radix samples. The results indicate that the established HPLC/UV method is suitable for quantitative analysis. PMID:23877237

  2. HPLC method to characterize cyanogen bromide collagen fractions containing pyridinoline groups.

    PubMed

    Bruno, R; Mazza, R; Calafiori, A R; Covello, C; Falbo, L; Martino, G; Marotta, M

    1997-01-01

    The HPLC method here described allows to separate CNBr collagen peptides within 2.5 h by reversed phase and gradient elution. The method is useful to determine both peptide bond and pyridinoline groups by absorbance spectophotometry. The fractions can be recovered and then submitted to other characterization techniques. PMID:10327703

  3. [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

  4. A computational study of multiple surface-directed phase separation in polymer blends under a temperature gradient

    NASA Astrophysics Data System (ADS)

    Tabatabaieyazdi, Mohammad; Chan, Philip K.; Wu, Jiangning

    2015-10-01

    The surface-directed phase separation (SDPS) phenomena of a model binary polymer blend quenched into the unstable region of its binary symmetric upper critical solution temperature phase diagram is numerically investigated using a mathematical model composed of the nonlinear Cahn-Hilliard (CH) theory for phase separation along with the Flory-Huggins-de Gennes (FHdG) free energy functional. The SDPS occurs in a square domain with a linear temperature gradient along the horizontal direction and with all sides having short range surface potential h 1. The effects of different quench depth, diffusion coefficient, surface potential, and temperature gradient were studied numerically. The numerical results indicate that there is a simultaneous competition between the four surfaces in attracting the preferred polymer. The side with a higher surface potential would win the competition against the side with a lower surface attraction in the case of a uniform quench. The numerical results also indicated a later transition time for higher values of h 1. As surface potential increased, the transition time from complete wetting to partial wetting occurred at a later time on the surface. The impact of different temperature gradient ΔT*/Δx* values on the surface enrichment rate with fixed temperature {{T}1}* at one surface and higher temperature {{T}2}* at the opposite surface was studied for the first time within a multiple surface potential set up. The results showed that higher values of ΔT*/Δx* increased the growth rate of the preferred polymer on the surface adding to the thickness of the wetting layer. The transition time from complete wetting to partial wetting occurred slightly later at the lower temperature side.

  5. Enantioselective separation and determination of adrafinil and modafinil on Chiralcel OJ-H column in rat serum and urine using solid-phase extraction followed by HPLC.

    PubMed

    Rao, R Nageswara; Shinde, Dhananjay D

    2009-08-01

    A simple and rapid normal-phase HPLC method for enantiospecific separation of a psychostimulant, adrafinil (ADL), and its metabolite modafinil (MDL) in rat serum and urine was developed. The separation was accomplished on a normal-phase polysaccharide stationary phase Chiralcel OJ-H using n-hexane-ethanol (62:38 v/v) as a mobile phase at a flow rate of 1.0 mL/min. Detection was carried out at 225 nm using a photo diode array (PDA) detector. The elution order of the enantiomers was determined by a polarimeter connected in series with the PDA. ADL and its metabolite were recovered from rat serum and urine by solid phase extraction using Oasis HLB cartridges and the mean recoveries were >or=80%. The enantiomers were eluted within 15 min without any interference from endogenous substances. The calibration curves were linear (r(2) > 0.998) in the concentration range of 1.20-500 microg/mL for ADL and MDL. The assay was specific, accurate, precise and reproducible (intra- and inter-day precisions RSDs <7.2%). ADL in rat serum was stable over three freeze-thaw cycles at ambient temperature for 4 h. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats. PMID:19353685

  6. Separation and detection of chloramphenicol with its nitro and amino analogues using HPLC equipped with reductive/oxidative electrochemical and ultraviolet detectors

    SciTech Connect

    Abou-Khalil, S.; Abou-Khalil, W.H.; Masoud, A.N.; Yunis, A.A.

    1987-05-01

    In their studies on chloramphenicol (CAP)-associated bone marrow injury, they have postulated that the p-nitro group of CAP undergoes nitro reduction yielding toxic and highly reactive derivatives. Elucidating the pathogenetic mechanism in CAP-induced injury requires specific and sensitive analytical tool to detect CAP metabolites that may be produced by mammalian cells. To accomplish their objective they have installed a uniquely suited HPLC system equipped with simultaneous dual detection: ultraviolet (UV) and electrochemical (EC). The system was constructed and specially designed to accommodate the use of the EC detector in both reductive and oxidative modes. Using this system, separation, quantitation (picomoles range) and identification of CAP were achieved with the following nitro and amino analogues: dehydrochloramphenicol, nitrophenylaminopropanedione, nitroso-chloramphenicol and aminochloramphenicol. Retention characteristics, hydrodynamic voltammograms under reductive and oxidative conditions and UV absorbance were determined. Applicability of the procedure to biological fluids was demonstrated by separation and detection of CAP after incubation with human blood. The data demonstrated that the reductive/oxidative EC modes in tandem with the conventional UV detector increased exponentially the selectivity and sensitivity of the system, and also provided a powerful basis for the identification of unknown metabolites.

  7. Rapid HPLC-MS method for the simultaneous determination of tea catechins and folates.

    PubMed

    Araya-Farias, Monica; Gaudreau, Alain; Rozoy, Elodie; Bazinet, Laurent

    2014-05-14

    An effective and rapid HPLC-MS method for the simultaneous separation of the eight most abundant tea catechins, gallic acid, and caffeine was developed. These compounds were rapidly separated within 9 min by a linear gradient elution using a Zorbax SB-C18 packed with sub 2 μm particles. This methodology did not require preparative and semipreparative HPLC steps. In fact, diluted tea samples can be easily analyzed using HPLC-MS as described in this study. The use of mass spectrometry detection for quantification of catechins ensured a higher specificity of the method. The percent relative standard deviation was generally lower than 4 and 7% for most of the compounds tested in tea drinks and tea extracts, respectively. Furthermore, the method provided excellent resolution for folate determination alone or in combination with catechins. To date, no HPLC method able to discriminate catechins and folates in a quick analysis has been reported in the literature. PMID:24734959

  8. 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. PMID:24296292

  9. Identification and quantification of coumarins in Peucedanum ostruthium (L.) Koch by HPLC-DAD and HPLC-DAD-MS.

    PubMed

    Vogl, Sylvia; Zehl, Martin; Picker, Paolo; Urban, Ernst; Wawrosch, Christoph; Reznicek, Gottfried; Saukel, Johannes; Kopp, Brigitte

    2011-05-11

    The rhizomes of Peucedanum ostruthium (L.) Koch (masterwort) are traditionally used in the alpine region as ingredient of liqueurs and bitters, and as a herbal drug. A sensitive and specific high-performance liquid chromatography-diode-array detection-mass spectrometry (HPLC-DAD-MS) method has been developed for the simultaneous identification and quantification of its main coumarins, oxypeucedanin hydrate, oxypeucedanin, ostruthol, imperatorin, osthole, isoimperatorin, and ostruthin. Fast HPLC separation could be achieved on an Acclaim C18 column (150 mm × 2.1 mm i.d., 3 μm) using a mobile phase gradient of acetonitrile-water modified with 0.01% acetic acid. The quantification by HPLC-DAD was performed with imperatorin as external standard and validated to demonstrate selectivity, linearity, precision, and accuracy. The content of the main coumarins was quantitated in various batches of commercial and field-collected rhizomes of Peucedanum ostruthium, as well as in beverages prepared thereof. PMID:21425828

  10. Iodixanol Gradient Centrifugation to Separate Components of the Low-Density Membrane Fraction from 3T3-L1 Adipocytes.

    PubMed

    Sadler, Jessica B A; Lamb, Christopher A; Gould, Gwyn W; Bryant, Nia J

    2016-02-01

    We optimized a set of fractionation techniques to facilitate the isolation of subcellular compartments containing insulin-sensitive glucose transporter isoform 4 (GLUT4), which is mobilized from GLUT4 storage vesicles (GSVs) in fat and muscle cells in response to insulin. In the absence of insulin, GLUT4 undergoes a continuous cycle of GSV formation and fusion with other compartments. Full membrane fractionation of 3T3-L1 adipocytes produces a low-density membrane fraction that contains both the constitutive recycling pool (the endosomal recycling compartments) and the insulin-sensitive pool (the GSVs). These two pools can be separated based on density using iodixanol gradient centrifugation, described here. PMID:26832683

  11. Subpopulations of MCF7 cells separate by Percoll gradient centrifugation: a model to analyze the heterogeneity of human breast cancer

    SciTech Connect

    Resnicoff, M.; Medrano, E.E.; Podhajcer, O.L.; Bravo, A.I.; Bover, L.; Mordoh, J.

    1987-10-01

    Exponentially growing MCF7 human breast cancer cells were separated in Percoll gradients into six different fractions of increasing density (A to F). These fractions could be subcultured and were found to contain different cellular subpopulations as defined by the following criteria: ability to generate other cellular subpopulations; growth rate; DNA synthesis; and expression of estrogen receptors, ras oncogene-encoded protein p21, and carcinoembryonic antigen. One of the minor fractions (E), which contained about 5% of the total cell number, appeared to contain the stem cells, on the basis of the following criteria: (i) its ability to reproduce the other cellular subpopulations, (ii) its high rate of growth and DNA synthesis, and (iii) the inability of the other subpopulations to generate it. The most differentiated subpopulation appeared to be the densest one (F), since it was the slowest growing and appeared to be the end point of the other subpopulations.

  12. Preparation of a novel porous poly (trimethylol propane triacrylate-co-ethylene dimethacrylate) monolithic column for highly efficient HPLC separations of small molecules.

    PubMed

    Bai, Xiaomei; Liu, Haiyan; Wei, Dan; Yang, Gengliang

    2014-02-01

    A novel poly (trimethylol propane triacrylate-co-ethylene dimethacrylate) [poly (TMPTA-co-EDMA)] monolith was prepared by in situ free-radical polymerization in a 50 mm × 4.6mm i.d. stainless steel column and was investigated for high performance liquid chromatography (HPLC). The porous structure of monolith was optimized by changing the conditions of polymerization. The chemical group of the monolithic column was confirmed by a Fourier transform infrared spectroscopy (FT-IR) method and the morphology of column structure was characterized by scanning electron microscopy (SEM). The mechanical strength and permeability were also studied. Finally, a series of low-molecular-weight organic compounds were utilized to evaluate the retention behaviors of the monolithic column. The result demonstrated that the prepared column exhibited an RP-chromatographic behavior and good separation performance. The method reproducibility was obtained by evaluating the run-to-run and column-to-column with relative standard deviations (RSDs) less than 0.7% (n=6) and 2.9% (n=6), respectively, which indicated that prepared monolithic columns had good reproducibility and stability. PMID:24401444

  13. Controllable Assembly and Separation of Colloidal Nanoparticles through a One-Tube Synthesis Based on Density Gradient Centrifugation.

    PubMed

    Qi, Xiaohan; Li, Minglin; Kuang, Yun; Wang, Cheng; Cai, Zhao; Zhang, Jin; You, Shusen; Yin, Meizhen; Wan, Pengbo; Luo, Liang; Sun, Xiaoming

    2015-05-01

    Self-assembly of gold nanoparticles into one-dimensional (1D) nanostructures with finite primary units was achieved by introducing a thin salt (NaCl) solution layer into density gradient before centrifugation. The electrostatic interactions between Au nanoparticles would be affected and cause 1D assembly upon passing through the salt layer. A negatively charged polymer such as poly(acrylic acid) was used as an encapsulation/stabilization layer to help the formation of 1D Au assemblies, which were subsequently sorted according to unit numbers at succeeding separation zones. A centrifugal field was introduced as the external field to overcome the random Brownian motion of NPs and benefit the assembly effect. Such a facile "one-tube synthesis" approach couples assembly and separation in one centrifuge tube by centrifuging once. The method can be tuned by changing the concentration of interference salt layer, encapsulation layer, and centrifugation rate. Furthermore, positively charged fluorescent polymers such as perylenediimide-poly(N,N-diethylaminoethyl methacrylate) could encapsulate the assemblies to give tunable fluorescence properties. PMID:25809533

  14. 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

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

    SciTech Connect

    Shi, Tujin; Fillmore, Thomas L.; Gao, Yuqian; Zhao, Rui; He, Jintang; Schepmoes, Athena A.; Nicora, Carrie D.; Wu, Chaochao; Chambers, Justin L.; Moore, Ronald J.; Kagan, Jacob; Srivastava, Sudhir; Liu, Alvin Y.; Rodland, Karin D.; Liu, Tao; Camp, David G.; Smith, Richard D.; Qian, Weijun

    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 of 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.

  16. 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.

  17. Development and application of a novel, dual-mode gradient, stability-indicating HPLC-DAD method for the simultaneous determination and purity assessment of mebeverine hydrochloride, diloxanide furoate and their corresponding major degradation products in combination with some gastrointestinal drugs in the form of oral doses.

    PubMed

    Mabrouk, Mokhtar; El-Fatatry, Hamed; Hewala, Ismail; Emam, Ehab

    2013-09-01

    A simple, precise, rapid, stability-indicating reversed phase high performance liquid chromatographic method with photodiode array detection was developed and validated for the determination of mebeverine hydrochloride in combination with sulpiride or with diloxanide furoate and metronidazole in the presence of their corresponding degradation products. Optimum separation was achieved in less than 10 min using an X-Bridge C18 column (150 mm × 4.6 mm i.d., 3.5 μm particle size); elution was accomplished via the application of a dual-mode solvent and flow rate gradient system. This elution system enables the separation of nine components within a cycle time of 15 min and with a resolution greater than 2.5. Detection was conducted at 230 nm, and purity assessment was performed using a photodiode array detector. The method has been validated with respect to specificity, linearity, accuracy, precision, limit of quantitation, limit of detection, robustness and ruggedness. The validation criteria were met in all cases. The developed HPLC method was successfully applied to commercial tablets. It was shown that this method is very sensitive to the determination of the degradation products, downward to 0.1 w/w% levels, which is far below the limits for testing these degradation products within their corresponding intact drugs. PMID:23770637

  18. 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

  19. 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.

  20. 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. PMID:3597643

  1. Separation of human thymocytes at different stages of maturation by centrifugation on a discontinuous gradient of colloidal silica gel.

    PubMed

    Goust, J M; Perry, L R

    1981-06-01

    Separation of human intrathymic cells on a discontinuous gradient of colloidal silica gel (Percoll) yielded four layers. The first (density 1.054 +/- 0.002 g/ml) contained stromal cells and a few thymocytes positive for terminal deoxynucleotidyl transferase (Tdt), most of which were bound to large Tdt-negative non-T cells. The second layer (1.069+/- 0.003 g/ml) contained large Tdt-negative thymocytes. The third and forth layers (1.075 +/-0.004 and 1.085 +/- 0.003 g/ml, respectively) contained smaller T cells, more than 95% of which were Tdt-positive. Functional studies revealed that cells from the first layer had a high level of spontaneous [3H]thymidine uptake but did not respond to lectins; the second layer responded to PHA, ConA, and allogeneic stimuli; and the third and fourth layers did not respond to lectin stimulation. Addition of cells from the first layer to the other layers at a 1 : 10 ratio significantly increased the mitogenic responses of the cells from the second layer, but not of those from the third or fourth layer. These results suggest that, as in mice and rats, low-density intrathymic thymocytes in humans represent more mature T cells, the percentage of which increases with age. PMID:6973841

  2. Microphase Separation and Shear Alignment of Gradient Copolymers: Melt Rheology and Small-Angle X-Ray Scattering Analysis

    SciTech Connect

    Mok, Michelle M.; Pujari, Saswati; Burghardt, Wesley R.; Dettmer, Christine M.; Nguyen, SonBinh T.; Ellison, Christopher J.; Torkelson, John M.

    2008-10-24

    The degree of microphase or nanophase segregation in gradient copolymers with compositions varying across the whole copolymer backbone is studied via low-amplitude oscillatory shear (LAOS) measurements and small-angle X-ray scattering (SAXS). Studies are done as a function of comonomer segregation strength, molecular weight (MW), gradient architecture and temperature. Controlled radical polymerization is used to synthesize strongly segregating styrene/4-acetoxystyrene (S/AS) and the more weakly segregating S/n-butyl acrylate (S/nBA) gradient copolymers. Results are compared to those from S/AS and S/nBA random and block copolymers. The higher MW S/AS gradient copolymer exhibits LAOS behavior similar to the highly microphase segregated S/AS block copolymer, while the lower MW S/AS gradient copolymer exhibits complex, nonterminal behavior indicative of a lower degree of microphase segregation. The S/nBA gradient copolymers demonstrate more liquidlike behavior, with the lower MW sample exhibiting near-Newtonian behavior, indicative of a weakly segregating structure, while the higher MW, steeper gradient sample shows behavior ranging from solidlike to more liquidlike with increasing temperature. With the exception of the lower MW S/nBA case, the gradient copolymers exhibit temperature-dependent LAOS behavior over a wide temperature range, reflecting their temperature-dependent nanodomain composition amplitudes. The S/AS samples have SAXS results consistent with the degree of microphase segregation observed via rheology. Shear alignment studies are done on the higher MW S/AS gradient copolymer, which is the most highly microphase segregated gradient copolymer. Rheology and SAXS provide evidence of shear alignment, despite the gradual variation in composition profile across the nanodomains of such gradient copolymers. A short review of the nomenclature and behavior of linear copolymer architectures is also provided.

  3. HPLC separation and FT-IR isomer differentiation of the 1,2,4,7/1,2,4,8-tetrachlorodibenzodioxin isomer pair: A theoretical/empirical approach to dibenzodioxin isomer assignmentt

    SciTech Connect

    Grainger, J.; Barnhart, E.; Patterson D.G. Jr.; Presser, D.

    1988-02-01

    The 1,2,4,7-and 1,2,4,8-tetrachlorodibenzodioxin (TCDD) isomers were separated by reversed-phase high-performance liquid chromatography (HPLC) with the use of a pyrene column. Fourier transform infrared (FT-IR) matrix isolation and vapor-phase spectra of the individual isomers were recorded. The spectra of the HPLC-seprated isomers correlate well with spectral subtraction results and were found to be distinct in three spectral regions: one of which allows for isomer structural assignment. Ambiguities and differences in published TCDD isomer FT-IR assignments are discussed in terms of a qualitative valence-bond approach and empirically derived estimates of ether linkage asymmetric stretching frequencies.

  4. 32 P-POSTLABELING AND HPLC SEPARATION OF DNA ADDUCTS FORMED BYDIESEL EXHAUST EXTRACTS IN VITRO AND IN MOUSE SKIN AND LUNG AFTERTOPICAL TREATMENT

    EPA Science Inventory

    Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form PAH- and nitrated-PAH-DNA adducts in rodent skin and lung. he aim of this study was to characterize by "P-postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro...

  5. Differentiation of tannin-containing herbal drugs by HPLC fingerprints.

    PubMed

    He, Yu; Wu, Qiaofeng; Hansen, S H; Cornett, C; Møller, C; Lai, Pingfan

    2013-03-01

    A new HPLC system coupled with multiple detectors - Diode array detector (DAD), fluorescence detector (FLD), electrochemical amperometric detector (ADC) and mass spectrometry detector (MSD) was developed for the characterization and differentiation of tannin-containing herbal drugs included in The European Pharmacopoeia. The HPLC separation system consisted of an Agilent ZORBAX Eclipse XDB C18 column and a gradient water and methanol as the mobile phase which was kept at a flow rate of 0.3 mL x min(-1). Four kinds of detectors were connected by a micro-splitter valve and simultaneously recorded the response of each analytical sample. Thirty-one samples from eight kinds of tannin-containing drugs were measured using this HPLC system and their signals from all detectors were comprehensively processed via principal component analysis (PCA). The statistic result demonstrates that thirty-one batches from different herbal drugs can be reasonably identified and systematically classified by their chemical fingerprints. The proposed multi-detector HPLC method aided by chemometrics not only offers a new pattern for the study of tannin-containing herbs, but also provides a useful foundation for quality control of herbal medicines. PMID:23556331

  6. Preparation of a novel ionic hybrid stationary phase by non-covalent functionalization of single-walled carbon nanotubes with amino-derivatized silica gel for fast HPLC separation of aromatic compounds.

    PubMed

    Aral, Hayriye; Çelik, K Serdar; Aral, Tarık; Topal, Giray

    2016-03-01

    Single-walled carbon nanotubes (SWCNTs) were immobilized on spherical silica gel with a 4-μm average particle size and a 60-Å average pore size. The amino-derivatized silica gel was non-covalently coated with carboxylated SWCNTs to preserve the structure of the nanotubes and their physico-chemical properties. The novel ionic hybrid stationary phase was characterized by scanning electron microscopy (SEM), infra-red (IR) spectroscopy and elemental analysis, and then, it was used to fill an empty 150×4.6mm(2) high-performance liquid chromatography (HPLC) column. Chromatographic parameters, such as the theoretical plate number, retention factor and peak asymmetry factor, and analytical parameters, such as the limit of detection (LOD), limit of quantification (LOQ), linear range, calibration equation, and R(2) value, and quantitative analysis parameters were calculated for all of the analytes. Using different mobile phases, five different classes of aromatic hydrocarbons were separated in a very short analysis time of 4-8min. Furthermore, a high theoretical plate number (up to 25000) and an excellent peak asymmetry factor (1.0) were obtained. The results showed that the surface of the SWNTs had very strong interactions with aromatic groups, therefore providing high selectivity for the separation of different classes of aromatic compounds. This study indicates that SWCNTs enable the extension of the application range of the newly prepared stationary phases for the fast separation of aromatic compounds by HPLC. PMID:26717810

  7. [HPLC fingerprint of liuwei dihuang soft capsule].

    PubMed

    Shi, Wei; Li, Jia-Chun; Yang, Su-De; Li, Yun; Jin, Rui-Ting; Sun, Xian-Ling; Wang, Zhen-Zhong; Bi, Yu-An; Xiao, Wei

    2014-12-01

    In order to establish HPLC fingerprint of Liuwei Dihuang soft capsule, and to provide certain reference for an quality control of it, the HPLC method was performed on an Agilent C18 (4.6 mm x 250 mm, 5 μm) column with acetonitrile-0.02% trifluoroacetic acid as mobile phase, gradient elution volume flow of 1.0 mL x min(-1), column temperature was 30 degrees C, detection wavelength: 0-60 min, 238 nm, 60-70 min, 210 nm. The software for chromatographic fingerprint was applied to analysis different batches of Liuwei Dihuang soft capsule samples. Sixteen mutual peaks were selected as the fingerprint peaks in 12 samples with loganin as the reference peak, and all of the detected peaks were separated effectively. Cluster analysis (HCA) and similarity analysis (SA) were done based on data of 12 samples clustering analysis of 12 batches of samples were divided into 2 categories. Including 7 for the first class, the rest was second, similarities calculated by SA were all above 0.92, indicating a good similarity between the reference and twelve batches of samples, also, the analysis results of HCA and SA basically the same. This method is simple with good precision, repeatability and stability, and provides the basis for Liuwei Dihuang soft capsule quality control. PMID:25911813

  8. 32P-postlabeling and HPLC separation of DNA adducts formed by diesel exhaust extracts in vitro and in mouse skin and lung after topical treatment.

    PubMed

    Savela, K; King, L; Gallagher, J; Lewtas, J

    1995-09-01

    Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form polycyclic aromatic hydrocarbon (PAH)- and nitrated PAH-DNA adducts in rodent skin and lung. The aim of this study was to characterize by 32P-postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro and in vivo by diesel extracts. The diesel particle extracts had known concentrations of benzo[a]pyrene, benzo[b,j,k]-fluoranthenes (B[b,j,k]F) and chrysene. DNA adducts were analyzed in calf thymus DNA incubated in vitro with PAHs activated by S9 mix and in skin and lung DNA from topically treated mice. The main diesel-derived DNA adduct formed in vitro and in vivo did not co-migrate on HPLC and large TLC plates with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE)-, B[b]F-,B[j]F-,B[k]F-or chrysene-DNA adduct standards. By co-chromatography DNA adducts formed by chrysene from both in vitro and in vivo samples were identified. Nissan diesel extract containing higher PAH concentrations than Volkswagen automobile extract formed skin DNA adducts that co-migrated with chrysene- and anti BPDE- DNA-derived adducts. We conclude that the use of a highly sensitive 32P-postlabeling method combined with HPLC improves the identification of PAH adducts formed by complex mixtures such as diesel exhaust extracts. PMID:7554058

  9. Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+-dependent DNA ligase inhibitor

    PubMed Central

    Zhou, Xiaofeng; Cai, Guoqiang; He, Yi; Tong, Guotong

    2016-01-01

    Cordycepin exhibits various bio-activities, including anticancer, antibacterial, antiviral and immune regulation activities, and is a significant focus of research. However, the preparation of high-purity cordycepin remains challenging. Also, the molecular target with which cordycepin interacts to cause an antibacterial effect remains unknown. In the present study, cordycepin was prepared by preparative high-performance liquid chromatography (prep-HPLC) and the purity obtained was 99.6%, indicating that this technique may be useful for the large-scale isolation of cordycepin in the future. The results of computational molecular docking analysis indicated that the interaction energy between cordycepin and NAD+-dependent DNA ligase (LigA) was lower than that between cordycepin and other common antibacterial targets. The highly pure cordycepin obtained by prep-HPLC demonstrated inhibitory activity against LigA from various bacteria in vitro. In conclusion, cordycepin may be useful as a broad-spectrum antibiotic targeting LigA in various bacteria. PMID:27588098

  10. 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. PMID:26549715

  11. Separation of the Herpesvirus Deoxyribonucleic Acid Duplex into Unique Fragments and Intact Strand on Sedimentation in Alkaline Gradients

    PubMed Central

    Frenkel, Niza; Roizman, Bernard

    1972-01-01

    Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 × 106 daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 × 106 to 39 × 106 daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with 3H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected

  12. Optimization of a CUPRAC-Based HPLC Postcolumn Assay and Its Applications for Ginkgo biloba L. Extracts

    PubMed Central

    Rimkiene, Laura; Ivanauskas, Liudas; Kubiliene, Asta; Vitkevicius, Konradas; Kiliuviene, Guoda; Jakstas, Valdas

    2015-01-01

    The aim of the present work was to improve and validate the HPLC-CUPRAC postcolumn method for the evaluation of active antioxidant markers from the acetonic extracts of Ginkgo biloba leaves. Improvement of the HPLC online assay was performed by evaluating the suitable loop temperature, the reaction loop length, and the impact of flow rate. Separation of the analytes was performed by the HPLC method on an ACE C18 analytical column using a gradient elution program. The separated antioxidant markers in the extracts reacted with copper(II)-neocuproine (Cu(II)-Nc) reagent in the postcolumn reaction coil. The reagent was reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate with a maximum absorption at 450 nm. Validation experiments confirmed sufficient precision, sensitivity, and effectiveness of the corresponding method, which could be used for further evaluations of active antioxidant compounds in similar plant materials. PMID:26236538

  13. Determination of the major constituents in fruit of Arctium lappa L. by matrix solid-phase dispersion extraction coupled with HPLC separation and fluorescence detection.

    PubMed

    Liu, He; Zhang, Yupu; Sun, Yantao; Wang, Xue; Zhai, Yujuan; Sun, Ye; Sun, Shuo; Yu, Aimin; Zhang, Hanqi; Wang, Yinghua

    2010-10-15

    The arctiin and arctigenin in the fruit of Arctium lappa L. were extracted by matrix solid-phase dispersion (MSPD) and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The experimental conditions for the MSPD were optimized. Silica gel was selected as dispersion adsorbent and methanol as elution solvent. The calibration curve showed good relationship (r>0.9998) in the concentration range of 0.010-5.0μgmL(-1) for arctiin and 0.025-7.5μgmL(-1) for arctigenin. The recoveries were between 74.4% and 100%. The proposed method consumed less sample, time and solvent compared with conventional methods, including ultrasonic and Soxhlet extraction. PMID:20810325

  14. Separating natural from anthropogenic causes of impairment in Zebra mussel (Dreissena polymorpha) populations living across a pollution gradient.

    PubMed

    Faria, Melissa; Ochoa, Victoria; Blázquez, Mercedes; Juan, Maria Fernandes San; Lazzara, Raimondo; Lacorte, Silvia; Soares, Amadeu M V M; Barata, Carlos

    2014-07-01

    The relationship between the reproductive stage, the total lipid content and eight broadly used biochemical stress responses were used to assess seasonal and pollutant effects across eleven different zebra mussel (Dreissena polymorpha) populations from the Ebro and Mijares river basin, Spain. Biochemical markers included superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione S transferase (GST), multixenobiotic transporter activity (MXR), lactate dehydrogenase (LDH), lipid peroxidation (LPO) and single strand DNA breaks. Principal component analyses of zebra mussel responses across an annual cycle, showed a marked gonad stage component in total lipid content and biochemical responses. The same response pattern was observed across the populations sampled along a broad geographical and pollution gradient. Population differences on the gonad developmental stage were highly correlated with most of the measured responses and unrelated with the pollution gradient. Conversely, bioaccumulation of organic and inorganic contaminant residues was more related to pollution sources than with the reproductive cycle. These results indicate that the reproductive cycle is the major factor affecting the temporal and spatial variation of the studied markers in D. polymorpha. PMID:24742819

  15. Effect and mechanism of a High Gradient Magnetic Separation (HGMS) and Ultraviolet (UV) composite process on the inactivation of microbes in ballast water.

    PubMed

    Ren, Zhijun; Zhang, Lin; Shi, Yue; Leng, Xiaodong; Shao, Jingchao

    2016-07-15

    The patented technology of a High Gradient Magnetic Separation (HGMS)-Ultraviolet (UV) composite process was used to treat ballast water. Staphylococcus aureus (S. aureus) was selected as the reference bacteria. After treatment by the HGMS-UV process, the concentration of S. aureus on the log 10 scale was lower than 2 at different flow rates, S. aureus suffered the most serious damage, and K(+) leakage of the bacteria was 1.73mg/L higher than separate 60min UV irradiation (1.17mg/L) and HGMS (0.12mg/L) processes. These results demonstrated that the HGMS-UV composite process was an effective approach to treat ballast water. Further, the HGMS process had synergistic action on the subsequent UV irradiation process and accelerated cell membrane damage. Meanwhile, the results of superoxide dismutase (SOD) activities of bacteria and DNA band analyses indicated that the inactivation mechanisms were different for HGMS and UV irradiation. PMID:27126180

  16. A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

    DOE PAGESBeta

    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. Ultimately, 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

  17. A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections.

    PubMed

    Moen, Scott T; Hatcher, Christopher L; Singh, Anup K

    2016-01-01

    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. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing. PMID:27054764

  18. A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

    PubMed Central

    Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

    2016-01-01

    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. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing. PMID:27054764

  19. Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: monocyte depletion depending upon activation of membrane potassium channels.

    PubMed

    Bøyum, A; Brincker Fjerdingstad, H; Martinsen, I; Lea, T; Løvhaug, D

    2002-07-01

    Routine one-step centrifugation procedures (Lymphoprep = LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions. After centrifugation the mononuclear cells (MNC = monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom. We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP = iohexol), a nonionic X-ray contrast agent. With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top. In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e. approximately 70-85% lymphocytes and 30-15% monocytes in the top fraction. The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl2 at room temperature. Monocyte depletion could not be obtained with LP. Under optimal conditions (density 1.075 g/ml, osmolality 280-300 mOsm/kg), the monocyte admixture amounted to approximately 1 (0-2)%, in separations with buffy coat samples. For freshly drawn blood, it was necessary to slightly modify the NP solution. The monocyte depletion was counteracted by blockers of K+ channels or by KCl in the cell suspension. Following incubation in NP of Percoll-separated cells, an enhanced release of K+ was observed. The results are interpreted as follows: NP mediates the opening of K+ channels of MNC, which leads to efflux of K+, accompanied with associated anions (Cl-). This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium. In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density

  20. Separation of single-walled carbon nanotubes by gel-based chromatography using surfactant step-gradient techniques and development of new instrumentation for studying SWCNT reaction processes

    NASA Astrophysics Data System (ADS)

    Breindel, Leonard M.

    Single-walled carbon nanotube (SWCNT) synthesis methods such as CoMoCATTM, HiPcoTM, pulsed laser vaporization (PLV), and catalytic chemical vapor deposition (CCVD) produce several different distributions of (n,m) SWCNT structures, where ( n,m) defines the nanotube diameter and chiral wrapping angle. Post-synthesis processing such as functionalization and/or separations must therefore be employed to yield high purity electronic or single (n,m) samples. Through the use of a surfactant gradient across a gel-based chromatographic column, separations of single (n,m) species can be achieved. Anionic surfactants such as SDS, SDBS, and AOT display different separation effectiveness for single (n,m) species. Results of near-infrared optical absorption for separated SWCNT surfactant suspensions will be discussed, leading to a broader understanding of the important factors necessary for the gel chromatography separation technique. In particular, the effects of SWCNT/surfactant micelle structure are found to be key to achieving fast, simple SWCNT electronic type separations. Additionally, development of new instrumentation for the near-infrared spectrofluorimetric analysis (NIR-SFA) of SWCNTs is useful to the advancement of fundamental SWCNT research and applications. NIR-SFA, for instance, allows for the (n,m) structures of a sample to be identified and monitored during the progress of a chemical reaction or separation experiment. Seeking to achieve the time resolutions necessary for such experiments, the design and optimizations of a system utilizing single-wavelength excitation by diode lasers coupled with a fast NIR detection system are presented.

  1. 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. PMID:27435687

  2. 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. PMID:25059196

  3. Separation and pre-concentration of glucocorticoids in water samples by ionic liquid supported vortex-assisted synergic microextraction and HPLC determination.

    PubMed

    Qin, Hui; Li, Bi; Liu, Mou Sheng; Yang, Ya Ling

    2013-04-01

    We have developed a synergic microextraction procedure based on ionic liquid for the pre-concentration and determination of glucocorticoids in water samples. Using nonionic surfactant Triton X-100 (TX-100) as synergic reagent, 1-butyl-3-methylimidazolium hexa-fluorophosphate accomplished extraction rapidly without heating in water bath. One key property of ionic liquids that highlights their potential is their wide liquid temperature range. The improved extraction was named as ionic liquid supported vortex-assisted synergic microextraction. Compared with the traditional liquid-liquid extraction and cloud point extraction, ionic liquid supported vortex-assisted synergic microextraction was accomplished in 8 min with considerably high recovery. The proposed method greatly improved the sensitivity of HPLC for the determination of glucocorticoids. The results obtained indicated a good linearity with the correlation coefficient of 0.997 over the range of 0.6-300 ng/mL and high sensitivity with LODs of 4.11, 9.19, and 7.50 ng/mL for hydrocortisone butyrate, beclomethasone dipropionate, and nandrolone phenylpropionate, respectively. The RSD of the method was 1.57-1.81% (n = 6) with enrichment factor of 99.85, and good recovery (≥97.24%). The method was successfully applied to the determination of glucocorticoids in mineral water, water of Dianchi lake, and tap water samples. PMID:23418157

  4. 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

  5. [Determination of amanitatoxins by HPLC].

    PubMed

    Nishizawa, Chiemi; Yamaura, Yoshio

    2003-10-01

    High-performance liquid chromatographic (HPLC) assay has been developed for the simultaneous determination of alpha-amanitin, beta-amanitin and phalloidin in serum. Three toxins were extracted by reflux in a water bath at 80 degrees C for one hour and purified by Sep-Pak Plus tC18 cartridges. The HPLC assay was performed under gradient conditions using Develosil RP AQUEOUS column. The moble phase consisted with a mixture of acetonitorile containing 0.01 M ammonium acetate(pH 5.0). The column effluence was monitored at 295 nm, 302 nm and 230 nm for 35 min. Detection limit of three toxins in serum were 0.2 microgram/ml respectively. High recovery yields in the range of 81.5-88.1% for toxins were obtained by using this method. PMID:14740566

  6. SEPARATION AND CHARACTERIZATION OF TETROL METABOLITES OF BENZO[A]PYRENE-DNA ADDUCTS USING HPLC AND SOLID-MATRIX ROOM TEMPERATURE LUMINESCENCE. (R824100)

    EPA Science Inventory

    Abstract

    Four tetrols of benzo[a]pyrene-DNA adducts were separated using reversed-phase high performance liquid chromatography. Chromatographic fractions containing a given tetrol were readily characterized with solid-matrix room temperature luminescence techniques. So...

  7. Chiral separation of cathinone derivatives used as recreational drugs by HPLC-UV using a CHIRALPAK® AS-H column as stationary phase.

    PubMed

    Mohr, Stefan; Taschwer, Magdalena; Schmid, Martin G

    2012-06-01

    Cathinone derivatives gained high popularity on the recreational drugs market during the past 10 years. All these compounds are chiral, and the pharmacological potency of the enantiomers of these stimulants is supposed to differ. The goal of this research was to develop a reliable and easy-to-perform high-performance liquid chromatography ultraviolet method for the chiral separation of a set of 24 cathinone derivatives. A commercially available CHIRALPAK® AS-H column consisting of amylose tris [(S)-α-methylbenzylcarbamate] coated on 5-µm silica gel was found to be suitable to resolve a majority of the tested compounds. High-performance liquid chromatography measurements were performed in normal phase mode under isocratic conditions with a mobile phase consisting of hexane, isopropanol, and triethylamine at a flowrate of 1 ml/min. The ratio between hexane and isopropanol was optimized by means of three model substances. Under final conditions with a mobile phase of hexane, isopropanol, and triethylamine (97:3:0.1), 19 out of 24 compounds were successfully resolved into their enantiomers and detected at a wavelength of 254 nm. A correlation between the substituents of the nitrogen atom and the separation results are shown. Furthermore, enantiomer separation results of four cathinone derivatives were compared with the results of their amphetamine analogs. PMID:22544697

  8. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods.

    PubMed

    Greening, David W; Xu, Rong; Ji, Hong; Tauro, Bow J; Simpson, Richard J

    2015-01-01

    Exosomes are 40-150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40-150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers). PMID:25820723

  9. 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. PMID:24310099

  10. Improving peak shapes with counter gradients in two-dimensional high performance liquid chromatography.

    PubMed

    Stevenson, Paul G; Bassanese, Danielle N; Conlan, Xavier A; Barnett, Neil W

    2014-04-11

    To achieve the greatest peak capacity in two-dimensional high performance liquid chromatography (2D-HPLC) a gradient should be operated in both separation dimensions. However, it is known that when an injection solvent that is stronger than the initial mobile phase composition is deleterious to peak performance, thus causing problems when cutting a portion from one gradient into another. This was overcome when coupling hydrophilic interaction with reversed phase chromatography by introducing a counter gradient that changed the solvent strength of the second dimension injection. It was found that an injection solvent composition of 20% acetonitrile in water gave acceptable results in one-dimensional simulations with an initial composition of 5% acetonitrile. When this was transferred to a 2D-HPLC separation of standards it was found that a marked improvement in peak shape was gained for the moderately retained analytes (phenol and dimethyl phthalate), some improvement for the weakly retained caffeine and very little change for the strongly retained n-propylbenzene and anthracene which already displayed good chromatographic profiles. This effect was transferred when applied to a 2D-HPLC separation of a coffee extract where the indecipherable retention profile was transformed to a successful application multidimensional chromatography with peaks occupying 71% of the separation space according to the geometric approach to factor analysis. PMID:24636564

  11. 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.

  12. 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.

  13. Analyses of procyanidins in foods using Diol phase HPLC

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Simultaneous separation/enrichment and detection of trace ciprofloxacin and lomefloxacin in food samples using thermosensitive smart polymers aqueous two-phase flotation system combined with HPLC.

    PubMed

    Lu, Yang; Chen, Bo; Yu, Miao; Han, Juan; Wang, Yun; Tan, Zhenjiang; Yan, Yongsheng

    2016-11-01

    Smart polymer aqueous two phase flotation system (SPATPF) is a new separation and enrichment technology that integrated the advantages of the three technologies, i.e., aqueous two phase system, smart polymer and flotation sublation. Ethylene oxide and propylene oxide copolymer (EOPO)-(NH4)2SO4 SPATPF is a pretreatment technique, and it is coupled with high-performance liquid chromatography to analyze the trace ciprofloxacin and lomefloxacin in real food samples. The optimized conditions of experiment were determined in the multi-factor experiment by using response surface methodology. The flotation efficiency of lomefloxacin and ciprofloxacin was 94.50% and 98.23% under the optimized conditions. The recycling experimentsshowed that the smart polymer EOPO could use repeatedly, which will reduce the cost in the future application. PMID:27211613

  15. Comprehensive off-line, two-dimensional liquid chromotography. Application to the separation of peptide digest

    SciTech Connect

    Marchetti, Nicola; Guiochon, Georges A

    2008-01-01

    The separation of the peptide digests of myoglobin and bovine serum albumin was performed with an off-line combination of two commercial, conventional HPLC columns. The first column was packed with a strong ion exchanger and eluted with a KCl gradient. The second column was packed with particles of C{sub 18}-bonded silica and eluted with an acetonitrile gradient. The conditional peak capacities of the 2D separations achieved exceed 7000 under the experimental conditions investigated. This performance is achieved at the cost of an analysis time of the order of 28 hours. Possible improvements to the separation method described here are discussed.

  16. Exploring the limit of accuracy for density functionals based on the generalized gradient approximation: Local, global hybrid, and range-separated hybrid functionals with and without dispersion corrections

    NASA Astrophysics Data System (ADS)

    Mardirossian, Narbe; Head-Gordon, Martin

    2014-05-01

    The limit of accuracy for semi-empirical generalized gradient approximation (GGA) density functionals is explored by parameterizing a variety of local, global hybrid, and range-separated hybrid functionals. The training methodology employed differs from conventional approaches in 2 main ways: (1) Instead of uniformly truncating the exchange, same-spin correlation, and opposite-spin correlation functional inhomogeneity correction factors, all possible fits up to fourth order are considered, and (2) Instead of selecting the optimal functionals based solely on their training set performance, the fits are validated on an independent test set and ranked based on their overall performance on the training and test sets. The 3 different methods of accounting for exchange are trained both with and without dispersion corrections (DFT-D2 and VV10), resulting in a total of 491 508 candidate functionals. For each of the 9 functional classes considered, the results illustrate the trade-off between improved training set performance and diminished transferability. Since all 491 508 functionals are uniformly trained and tested, this methodology allows the relative strengths of each type of functional to be consistently compared and contrasted. The range-separated hybrid GGA functional paired with the VV10 nonlocal correlation functional emerges as the most accurate form for the present training and test sets, which span thermochemical energy differences, reaction barriers, and intermolecular interactions involving lighter main group elements.

  17. Exploring the limit of accuracy for density functionals based on the generalized gradient approximation: Local, global hybrid, and range-separated hybrid functionals with and without dispersion corrections

    SciTech Connect

    Mardirossian, Narbe; Head-Gordon, Martin

    2014-05-14

    The limit of accuracy for semi-empirical generalized gradient approximation (GGA) density functionals is explored by parameterizing a variety of local, global hybrid, and range-separated hybrid functionals. The training methodology employed differs from conventional approaches in 2 main ways: (1) Instead of uniformly truncating the exchange, same-spin correlation, and opposite-spin correlation functional inhomogeneity correction factors, all possible fits up to fourth order are considered, and (2) Instead of selecting the optimal functionals based solely on their training set performance, the fits are validated on an independent test set and ranked based on their overall performance on the training and test sets. The 3 different methods of accounting for exchange are trained both with and without dispersion corrections (DFT-D2 and VV10), resulting in a total of 491 508 candidate functionals. For each of the 9 functional classes considered, the results illustrate the trade-off between improved training set performance and diminished transferability. Since all 491 508 functionals are uniformly trained and tested, this methodology allows the relative strengths of each type of functional to be consistently compared and contrasted. The range-separated hybrid GGA functional paired with the VV10 nonlocal correlation functional emerges as the most accurate form for the present training and test sets, which span thermochemical energy differences, reaction barriers, and intermolecular interactions involving lighter main group elements.

  18. Separation system suitability (3S): a new criterion of chromatogram classification in HPLC based on cross-evaluation of separation capacity/peak symmetry and its application to complex mixtures of anthraquinones.

    PubMed

    Nowik, Witold; Héron, Sylvie; Bonose, Myriam; Tchapla, Alain

    2013-10-01

    A comparison of chromatograms obtained in a series of separation conditions for a given complex mixture may be done with a series of chromatographic descriptors. In this study, we used two descriptors: the number of critical pairs and symmetry of peaks, further rescaled and converted to the corresponding critical pairs' coefficient (CPc) and symmetry coefficient (Sc). Considering the difficulty of appreciating global separation quality using CPc and Sc criteria separately, as their respective values are usually uncorrelated, a double-criteria cross-evaluation system was required. For that purpose we tested the commonly used multi-criteria decision-making method - Derringer's desirability function (D) - as well as the recently introduced sum of ranking differences (SRD). To facilitate the graphical comparison of both approaches, the desirability function (D) was used in the inverse form (Dinv). The advantages and drawbacks of both evaluation methods, especially the respective under- or over-evaluation of outliers, caused us to introduce a new ranking approach, separation system suitability (3S). The obtained suitability rankings for the three tested approaches (Dinv, SRD and 3S) are different; nevertheless, 3S appears to be the most balanced and the easiest to interpret as well. The approach developed for selection of suitable systems was applied to the problem of separation of complex mixtures through the analysis of a series of standards of anthraquinone derivatives. To judge the pertinence of this evaluation, a sample containing a number of natural anthraquinones extracted from the bark of Indian mulberry (Morinda citrifolia) was analysed. In conclusion, the proposed methodology for the cross-evaluation of the series of chromatograms using single specific descriptors (CPc and Sc) through a global composite descriptor (3S) significantly simplifies the decision that separation systems are the most suitable for the separation of complex target mixtures of compounds

  19. Quantitative and qualitative HPLC analysis of thermogenic weight loss products.

    PubMed

    Schaneberg, B T; Khan, I A

    2004-11-01

    An HPLC qualitative and quantitative method of seven analytes (caffeine, ephedrine, forskolin, icariin, pseudoephedrine, synephrine, and yohimbine) in thermogenic weight loss preparations available on the market is described in this paper. After 45 min the seven analytes were separated and detected in the acetonitrile: water (80:20) extract. The method uses a Waters XTerra RP18 (5 microm particle size) column as the stationary phase, a gradient mobile phase of water (5.0 mM SDS) and acetonitrile, and a UV detection of 210 nm. The correlation coefficients for the calibration curves and the recovery rates ranged from 0.994 to 0.999 and from 97.45% to 101.05%, respectively. The qualitative and quantitative results are discussed. PMID:15587578

  20. Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis.

    PubMed Central

    Rice, K G; Rottink, M K; Linhardt, R J

    1987-01-01

    Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed. Images Fig. 3. Fig. 5. Fig. 7. PMID:3446173

  1. Development of an HPLC post-column antioxidant assay for Solidago canadensis radical scavengers.

    PubMed

    Marksa, Mindaugas; Radušienė, Jolita; Jakštas, Valdas; Ivanauskas, Liudas; Marksienė, Rūta

    2016-01-01

    The aim of this work was to modify and validate the post-column high-performance liquid chromatography (HPLC)-ABTS and DPPH methods for evaluating the antioxidant activity of the methanolic extracts of Solidago canadensis (Canadian goldenrod) leaves and flowers. Separation of the analytes was performed via the HPLC-PDA method on a YMC analytical column using a gradient elution program. Three compounds with antioxidant properties - chlorogenic acid, rutin and isoquercitrin - and two unidentified antioxidants were established. The research showed that the coil temperature regimes and loop length combinations influence the optimised post-column assay method for detecting the antioxidant activity of goldenrod radical scavengers. Investigations established that the temperature in the reaction coil was a substantial factor contributing to the signal strength of the analytes after reacting with the DPPH and ABTS radicals. PMID:25835071

  2. The retention behavior of ginsenosides in HPLC and its application to quality assessment of Radix Ginseng.

    PubMed

    Hu, Ping; Luo, Guo-An; Wang, Qing; Zhao, Zhong-Zhen; Wang, Wan; Jiang, Zhi-Hong

    2008-10-01

    This study systematically investigated the retention behavior of seven neutral ginsenosides Rg(1), Re, Rf, Rb(1), Rb(2), Rc, Rd, and an acidic ginsenoside R(0), the major pharmacologically active components of Radix Ginseng with RP-HPLC. The effects of solvent, pH value, ionic strength of the mobile phase, and column temperature were investigated using an octadecylsiloxane-bonded silica gel column. Based on the ginsenosides' retention characteristics, the concentration of acetonitrile and the gradient of the mobile phase needed to maintain the baseline separation of the major neutral ginsenosides in Radix Ginseng were theoretically predicted. Furthermore, the ionic strength of mobile-phase necessary to achieve good resolution of the neutral ginsenosides and acidic ginsenosides was carefully investigated. According to the results of the quantitative analysis of ginsenosides in eight batches of ginseng samples from different sources, the developed HPLC technique may be a valuable tool for the quality assessment of Radix Ginseng. PMID:18958416

  3. The retention behavior of ginsenosides in HPLC and its application to quality assessment of Radix Ginseng.

    PubMed

    Hu, Ping; Luo, Guo-An; Wang, Qing; Zhao, Zhong-Zhen; Wang, Wan; Jiang, Zhi-Hong

    2009-05-01

    This study systematically investigated the retention behavior of seven neutral ginsenosides Rg(1), Re, Rf, Rb(1), Rb(2), Rc, Rd, and an acidic ginsenoside R(0), the major pharmacologically active components of Radix Ginseng with RP-HPLC. The effects of solvent, pH value, ionic strength of the mobile phase, and column temperature were investigated using an octadecylsiloxane-bonded silica gel column. Based on the ginsenosides' retention characteristics, the concentration of acetonitrile and the gradient of the mobile phase needed to maintain the baseline separation of the major neutral ginsenosides in Radix Ginseng were theoretically predicted. Furthermore, the ionic strength of mobile-phase necessary to achieve good resolution of the neutral ginsenosides and acidic ginsenosides was carefully investigated. According to the results of the quantitative analysis of ginsenosides in eight batches of ginseng samples from different sources, the developed HPLC technique may be a valuable tool for the quality assessment of Radix Ginseng. PMID:19471880

  4. Analysis of the monosaccharide composition of fucoidan by precolumn derivation HPLC

    NASA Astrophysics Data System (ADS)

    Zhang, Jingjing; Zhang, Quanbin; Wang, Jing; Shi, Xuelian; Zhang, Zhongshan

    2009-09-01

    We developed an HPLC method for analysis of the monosaccharide composition of fucoidans. The fucoidan was hydrolyzed into monosaccharides with 2 mol/L trifluoroacetic acid. Using ribose as the internal standard, the monosaccharide derivatives, obtained with 1-Phenyl-3-methyl-5-pyrazolone (PMP), were separated by reverse-phase HPLC using a gradient elution process, and monitored by ultraviolet detection at 245 nm. In the concentration range of 0.1-2.0 mmol/L, the peak area of each monosaccharide had a good linear relationship with its concentration ( r 2>0.998). The average recoveries of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, and fucose were 86.2%, 95.1%, 62.5%, 102.0%, 94.8%, 66.6%, and 105.1%, respectively. This method was accurate and had good reproducibility and could be used to determine the monosaccharide contents of fucoidans.

  5. Simultaneous quantitation of tobramycin and colistin sulphate by HPLC with evaporative light scattering detection.

    PubMed

    Clarot, I; Storme-Paris, I; Chaminade, P; Estevenon, O; Nicolas, A; Rieutord, A

    2009-08-15

    A rapid and simple method for the simultaneous determination of tobramycin and colistin sulphate in a pharmaceutical formulation by reversed phase HPLC and evaporative light scattering detection is described. Chromatographic separation was carried out in gradient mode using a Zorbax SB C18 column (150mmx4mm, 3.5microm) with mobile phases of acetonitrile and water containing trifluoroacetic at 1ml/min. The method was validated using methodology described by the International Conference of Harmonization. The method was shown to be specific, precise, accurate and linear. Real samples were analyzed to demonstrate the applicability of the chromatographic method in a routine use. PMID:19372021

  6. Solvent and temperature gradients in separation of synthetic oxyethylene-oxypropylene block (co)polymers using high-temperature liquid chromatography.

    PubMed

    Welerowicz, Tomasz; Jandera, Pavel; Novotná, Katerina; Buszewski, Boguslaw

    2006-05-01

    Chromatographic behavior of synthetic block (co)oligomer samples (EO)n(PO)m(EO)n and (PO)n(EO)m(PO)n with different distribution of propylene oxide (PO) and ethylene oxide (EO) monomer units was investigated on three types of stationary phases on zirconium dioxide support: Zr-PS (polystyrene), Zr-carbon, and Zr-carbon C18. The effects of the distribution and sequence of the oxyethylene and oxypropylene monomer units on the chromatographic retention depend on the type of the stationary phase, but are strongly affected by the organic modifier (methanol or ACN) in aqueous-organic mobile phase. Special attention was focused on the influence of the mobile-phase composition on the separation according to the EO and PO distribution. Zirconia-based columns are stable at elevated temperatures and can be used in high-temperature LC (HTLC); hence, we investigated the temperature effects on the chromatographic behavior up to 90 degrees C. The applications of solvent and temperature gradients were compared on the zirconia stationary phases in the RP mode. PMID:16830731

  7. 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.

  8. Determination of CMPO using HPLC -UV

    SciTech Connect

    Gracy Elias; Gary S. Groenewold; Bruce J. Mincher; Stephen P. Mezyk

    2012-06-01

    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 system precision. CMPO-nitric acid complexes were also identified, that account for the apparent loss of CMPO in acidic environment, independent of irradiation.

  9. 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. PMID:25572656

  10. The detection of radical scavenging compounds in crude extract of borage (Borago officinalis L.) by using an on-line HPLC-DPPH method.

    PubMed

    Bandoniene, Donata; Murkovic, Michael

    2002-01-01

    The rapid evaluation of antioxidant activity of crude borage (Borago officinalis L.) extract was determined by using DPPH free radical method. This borage extract resulted in a rapid decrease of the absorbance and showed very high hydrogen-donating capacity towards the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical. A new HPLC-DPPH on-line method was applied for a screening of several radical scavenging components in this borage extract as well as for quantitative analysis. This on-line HPLC-DPPH method was developed using a methanolic solution of DPPH-stable radical. The HPLC-separated analytes reacted post-column with the DPPH solution in methanol. The induced bleaching was detected as a negative peak photometrically at 515 nm. The separation of antioxidative components was carried out by gradient HPLC with mobile-phase composition ranging from 2% to 80% acetonitrile with 2% acetic acid in water, UV detection was carried out at 280 nm. The HPLC analysis of borage extract revealed the presence of several radical scavenging components in the borage extract. The results obtained from the chromatograms suggest that some compounds present in the extract possess high radical quenching ability. The dominant antioxidative compound in the crude extract of borage leaves was identified as rosmarinic acid. PMID:12406585

  11. 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. PMID:17079108

  12. Microfabricated refractive index gradient based detector for reversed-phase liquid chromatography with mobile phase gradient elution.

    PubMed

    McBrady, Adam D; Synovec, Robert E

    2006-02-10

    Typical refractive index (RI) detectors for liquid chromatography (LC) are not well suited to application with mobile phase gradient elution, due to the difficulty in correcting for the detected baseline shift during the gradient. We report a sensitive, highly reproducible, microfabricated refractive index gradient (micro-RIG) detector that performs well with mobile phase gradient elution LC. Since the micro-RIG signal remains on-scale throughout the mobile phase gradient, one can apply a baseline correction procedure. We demonstrate that by collecting two mobile phase gradient blanks and subtracting one of them from the other, a reproducible, flat baseline is achieved. Therefore, subtracting a blank from a separation provides a baseline corrected chromatogram with reasonably high signal-to-noise ratio for eluting analytes. The micro-RIG detector uses a collimated diode laser beam to optically probe a RIG formed perpendicular to the laminar flow direction within a microfabricated borosilicate glass chip. The chip-based design of the detector is suitable for either traditional bench-top or LC-on-a-chip technologies. We report reversed phase high performance liquid chromatography (RP-HPLC) separations of proteins and polymers, over mobile phase gradient conditions of 67% A:33% B to 3% A:97% B by volume, where A is 96% methanol:3.9% water:0.1% trifluoroacetic acid (TFA), and B is 3.9% methanol:96% water:0.1% TFA. The separations were performed on a Jupiter 5 mu C4 300 A 150 mm x 1.0 mm Phenomenex column at a flow rate of 20 microl/min. Viscosity changes during the mobile phase gradient separation are found to shift the on-chip merge position of the detected concentration gradient (i.e., RIG), in a reproducible fashion. However, this viscosity effect makes detection sensitivity vary throughout the mobile phase gradient, due to moving the optimized position of the probe beam in relation to the analyte concentration gradient being probed. None-the-less, consistent limits

  13. Determination of amphetamine by HPLC after acetylation.

    PubMed

    Veress, T

    2000-01-01

    An analytical procedure has been developed for the HPLC determination of amphetamine by off-line pre-column derivatization. The proposed procedure consists of sample preparation by acetylation of amphetamine with acetic anhydride and a subsequent reversed-phase HPLC separation on an octadecyl silica stationary phase with salt-free mobile phase (tetrahydrofuran, acetonitrile, 0.1% triethylamine in water, 15:15:70 v/v) applying UV-detection. The applicability of the elaborated procedure is demonstrated with results obtained by analysis of real samples seized in the Hungarian black market. PMID:10641931

  14. Development and Validation of HPLC and HPTLC Methods for Estimation of Glabridin in Extracts of Glycyrrhiza glabra.

    PubMed

    Viswanathan, Vivek; Mukne, Alka P

    2016-01-01

    Glabridin is a major bioactive phytoconstituent of licorice. This work discusses the development and validation of HPLC and HPTLC methods for analysis of glabridin in licorice. The HPLC separation was performed using a Purospher STAR RP-18e column (5 μm silica particle size, 250 mm × 4.6 mm inner diameter) with gradient elution of 0.2% acetic acid in water-acetonitrile. The flow rate was 1 mL/min. Quantification was performed at a detection wavelength of 280 nm. HTPLC separation was performed on precoated silica gel 60 F254 aluminum plate (10 × 10 cm, 250 μm thickness). A linear ascending development was done using a mobile phase of hexane-ethyl acetate-chloroform (5 + 4 + 3, v/v/v). After development, the plates were scanned at 285 nm. Both of the methods provided good separation of glabridin from other constituents of licorice extract. The methods were validated as per ICH guidelines. Comparison by Student t-test showed that there was a statistically insignificant difference between the mean glabridin content estimated by both methods at 95% confidence interval. The glabridin content in licorice extract was 3.90% by HPLC and 3.79% by HPTLC. PMID:27103104

  15. Analysis of Piperaceae germplasm by HPLC and LCMS: a method for isolating and identifying unsaturated amides from Piper spp extracts.

    PubMed

    Scott, Ian M; Puniani, Evaloni; Jensen, Helen; Livesey, John F; Poveda, Luis; Sanchez-Vindas, Pablo; Durst, Tony; Arnason, John T

    2005-03-23

    A method for extraction and high performance liquid chromatography-mass spectrometer (HPLC-MS) analysis of the medicinally important genus Piper (Piperaceae) was developed. This allows for a rapid and accurate measure of unsaturated amides, or piperamides, in black pepper, Piper nigrum L., and in wild species from Central America. Reflux extraction provided the highest recovery of piperine (>80%) from leaf and peppercorn material. HPLC analysis using a binary gradient of acetonitrile and water separated the major amide peaks between 5 and 12 min. Atmospheric pressure chemical ionization (APCI)-MS improved the detection limit to 0.2 ng, 10-fold below the 2 ng limit of the HPLC-diode array detector (DAD) based on linear standard curves between 0.1 and 250 microg/mL (R2 = 0.999). The HPLC-MS method identified pellitorine, piperylin, 4,5-dihydropiperlonguminine, piperlonguminine, 4,5-dihydropiperine, piperine, and pipercide. The biological activity of six Costa Rican Piper species assessed by mosquito larval bioassays correlated well with piperamide content. PMID:15769112

  16. Efficient HPLC method development using structure-based database search, physico-chemical prediction and chromatographic simulation.

    PubMed

    Wang, Lin; Zheng, Jinjian; Gong, Xiaoyi; Hartman, Robert; Antonucci, Vincent

    2015-02-01

    Development of a robust HPLC method for pharmaceutical analysis can be very challenging and time-consuming. In our laboratory, we have developed a new workflow leveraging ACD/Labs software tools to improve the performance of HPLC method development. First, we established ACD-based analytical method databases that can be searched by chemical structure similarity. By taking advantage of the existing knowledge of HPLC methods archived in the databases, one can find a good starting point for HPLC method development, or even reuse an existing method as is for a new project. Second, we used the software to predict compound physicochemical properties before running actual experiments to help select appropriate method conditions for targeted screening experiments. Finally, after selecting stationary and mobile phases, we used modeling software to simulate chromatographic separations for optimized temperature and gradient program. The optimized new method was then uploaded to internal databases as knowledge available to assist future method development efforts. Routine implementation of such standardized workflows has the potential to reduce the number of experiments required for method development and facilitate systematic and efficient development of faster, greener and more robust methods leading to greater productivity. In this article, we used Loratadine method development as an example to demonstrate efficient method development using this new workflow. PMID:25481084

  17. Stability-Indicating RP-HPLC Method for the Determination of Ambrisentan and Tadalafil in Pharmaceutical Dosage Form

    PubMed Central

    Patel, Jayvadan K.; Patel, Nilam K.

    2014-01-01

    Abstract A simple, rapid, and highly selective RP-HPLC method was developed for the simultaneous determination of Ambrisentan (AMB) and Tadalafil (TADA) drug substances in the fixed dosage strength of 10 mg and 40 mg, respectively. Effective chromatographic separation was achieved using a Hypersil GOLD C18 column (150 mm × 4.6 mm internal diameter, 5 μm particle size) with a mobile phase composed of methanol, water, and acetonitrile in the ratio of 40:40:20 (by volume). The mobile phase was pumped using a gradient HPLC system at a flow rate of 0.5 mL/min, and quantification of the analytes was based on measuring their peak areas at 260 nm. The retention times for Ambrisentan and Tadalafil were about 2.80 and 7.10 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Calibration curves were linear in the ranges of 1–20 μg/mL for Ambrisentan and 4–80 μg/mL for Tadalafil with correlation coefficients >0.990. The proposed method proved to be selective and stability-indicating by the resolution of the two analytes from the forced degradation (hydrolysis, oxidation, and photolysis) products. The validated HPLC method was successfully applied to the analysis of AMB and TADA in pharmaceutical dosage form. PMID:26279975

  18. Method development and validation for the analysis of a new anti-cancer infusion solution via HPLC.

    PubMed

    Donnarumma, Fabrizio; Schober, Margot; Greilberger, Joachim; Matzi, Veronika; Lindenmann, Jörg; Maier, Alfred; Herwig, Ralf; Wintersteiger, Reinhold

    2011-01-01

    A fast and simple HPLC method has been developed and validated for the quantification of a completely new anti-cancer drug during the manufacturing process. The combination of four compounds including α-ketoglutaric acid, hydroxymethylfurfural, N-acetyl-L-methionine and N-acetyl-L-selenomethionine, administered intravenously, is still in test phase but has already shown promising results in cancer therapy. HPLC separation was achieved on an RP-18 column with a gradient system. However, the highly different concentrations of the compounds required a variation in the detection wavelength within one run. In order to produce a chromatogram where peaks were comparable on a similar range scale, detection at absorption maxima for the two most concentrated components was avoided. After optimization of the gradient program it was possible to detect all four substances within 14 min in spite of their strongly different chemical structure. The method developed was validated for accuracy, repeatability, reproducibility and robustness in relation to temperature and pH of buffer. Linearity as well as the limit of detection and quantification were determined. This HPLC method was found to be precise, accurate and reproducible and can be easily used for in-line process control during the manufacture of the anti-tumour infusion solution. PMID:21246718

  19. 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. PMID:21360668

  20. [Establishment and application of HPLC-QAMS for quality evaluation of Chuanxiong Rhizoma].

    PubMed

    Qiao, Feng-xian; Cai, Hao; Tu, Peng-fei; Pei, Ke; Song, Xiao-qing

    2015-06-01

    A quantitative analysis method of multi-components with a single marker (QAMS) for simultaneous determination of six marker compounds (one from phenolic acids and five from phthalides) in Chuanxiong Rhizoma was established by applying HPLC and using butylidenephthalide as the internal reference substance. And also the feasibility and accuracy of the established method for quality evaluation and application of Chuanxiong Rhizoma were investigated and validated. The analysis was performed with the mobile phase consisting of acetonitrile - 0.2% aqueous formic acid. The flow rate was 1.0 mL . min-1 and the column temperature was maintained at 30 °C. The detection wavelengths were set at 252 nm (for ferulic acid, Z-ligustilide, and butylidenephthalide) and 266 nm (for senkyunolide I, senkyunolide A, and coniferyl ferulate), separately, and 20 µL was injected for analysis with gradient elution. The results showed that there were no significant differences observed between the HPLC-QAMS method and the external standard method (RSD <5%). The relative correction factors were credible (RSD < 5%) in changed chromatographic conditions. The established HPLC-QAMS method can be accurately used for simultaneously evaluating and controlling the quality of Chuanxiong Rhizoma with multi-components. PMID:26521448

  1. 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.

  2. Arsenic speciation in chinese seaweeds using HPLC-ICP-MS and HPLC-ES-MS.

    PubMed

    Van Hulle, Marijn; Zhang, Chao; Zhang, Xinrong; Cornelis, Rita

    2002-05-01

    Three common Chinese edible seaweeds, one brown (Laminaria japonica) and two red (Porphyra crispata and Eucheuma denticulatum), were examined for their total arsenic content. The As species were extracted with yields of 76.4, 69.8 and 25.0%, respectively. Anion-exchange and cation-exchange high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were used for the separation of the different arsenic species in two of the three seaweed extracts (Laminaria and Porphyra). The main arsenic species in the algal extracts are arseno sugars, although it has been shown that the Laminaria seaweed contains significant amounts of dimethylarsinic acid (DMA). HPLC was coupled with electrospray mass spectrometry (ES-MS) for structural confirmation of the arsenic species. The mass spectrometer settings for the arseno sugars were optimised using standards. The conclusions drawn on the basis of HPLC-ICP-MS were confirmed by the HPLC-ES-MS data. The HPLC-ES-MS method is capable of determining both arseno sugars and DMA in the seaweeds. The unknown compounds seen in the HPLC-ICP-MS chromatogram of Laminaria could not be ascribed to trimethylarsenic oxide or tetramethylarsonium ion. PMID:12081041

  3. HPLC analysis and purification of peptides.

    PubMed

    Mant, Colin T; Chen, Yuxin; Yan, Zhe; Popa, Traian V; Kovacs, James M; Mills, Janine B; Tripet, Brian P; Hodges, Robert S

    2007-01-01

    High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptides/proteins by size-exclusion chromatography and monitoring the hydrophilicity/hydrophobicity of amphipathic alpha-helical peptides, a vital precursor for the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins. PMID:18604941

  4. Purification, separation and extraction of inner tubes from double-walled carbon nanotubes by tailoring density gradient ultracentrifugation using optical probes

    PubMed Central

    Rohringer, Philip; Shi, Lei; Liu, Xianjie; Yanagi, Kazuhiro; Pichler, Thomas

    2014-01-01

    We studied the effect of varying sonication and centrifugation parameters on double-walled carbon nanotubes (DWCNT) by measuring optical absorption and photoluminescence (PL) of the samples. We found that by using a low sonication intensity before applying density gradient ultracentrifugation (DGU), only inner tube species with a diameter ⩽0.8 nm can be identified in absorption measurements. This is in stark contrast to the result after sonicating at higher intensities, where also bigger inner tubes can be found. Furthermore, by comparing PL properties of samples centrifugated either with or without a gradient medium, we found that applying DGU greatly enhances the PL intensity, whereas centrifugation at even higher speeds but without a gradient medium results in lower intensities. This can be explained by extraction of inner tubes from their host outer tubes in a two-stage process: the different shearing forces from the sonication treatments result in some DWCNT to be opened, whereas others stay uncut. A subsequent application of DGU leads to the extraction of the inner tubes or not if the host nanotube stayed uncut or no gradient medium was used. This work shows a pathway to avoid this phenomenon to unravel the intrinsic PL from inner tubes of DWCNT. PMID:25843961

  5. HPLC Neurotransmitter Analysis.

    PubMed

    Holm, Thomas Hellesøe; Isaksen, Toke Jost; Lykke-Hartmann, Karin

    2016-01-01

    High performance liquid chromatography (HPLC) is a powerful tool to measure neurotransmitter levels in specific tissue samples and dialysates from patients and animals. In this chapter, we list the current protocols used to measure neurotransmitters in the form of biogenic amines from murine brain samples. PMID:26695044

  6. Separated flow

    NASA Technical Reports Server (NTRS)

    Sellers, W. L., III; Dunham, R. E., Jr.; Goodman, W. L.; Howard, F. G.; Margason, R. J.; Rudy, D. H.; Rumsey, C. L.; Stough, H. P., III; Thomas, J. L.

    1986-01-01

    A brief overview of flow separation phenomena is provided. Langley has many active research programs in flow separation related areas. Three cases are presented which describe specific examples of flow separation research. In each example, a description of the fundamental fluid physics and the complexity of the flow field is presented along with a method of either reducing or controlling the extent of separation. The following examples are discussed: flow over a smooth surface with an adverse pressure gradient; flow over a surface with a geometric discontinuity; and flow with shock-boundary layer interactions. These results will show that improvements are being made in the understanding of flow separation and its control.

  7. Peculiarities of zone migration and band broadening in gradient reversed-phase high-performance liquid chromatography of proteins with respect to membrane chromatography.

    PubMed

    Belenkii, B G; Podkladenko, A M; Kurenbin, O I; Mal'tsev, V G; Nasledov, D G; Trushin, S A

    1993-08-13

    The peculiarities of zone migration and band broadening in the reversed-phase gradient HPLC of proteins were investigated. In the isocratic mode a critical composition of the mobile phase was found at which all proteins regardless of their molecular mass migrate with equal velocity and have a capacity factor equal to the phase ratio (VP/V0), i.e., the same capacity factor as a marker of total accessible volume would have in steric exclusion chromatography. It is shown that steric exclusion conditions are never achieved in gradient HPLC. In the first (adsorption stage) of gradient elution where the separation takes place the velocity of a protein increases until it becomes equal to the velocity of the desorbing solvent front at a critical distance X0 from column entrance. Strong broadening is characteristic of this stage. In the second (critical) stage the protein travels the remaining distance (L-X0) with the velocity of the solvent. A definition of X0 is given allowing one very simple calculation of the minimum permissible column length as a function of gradient steepness, mobile phase velocity and protein adsorption parameter. When x = X0 the protein zone has the smallest dispersion. Making L < X0 is especially disadvantageous, as it leads to anomalous bandspreading. The theory of gradient HPLC was refined on this basis and the usefulness of this approach in high-performance membrane chromatography is demonstrated. PMID:8408410

  8. Identification of Rhodiola species by using RP-HPLC*

    PubMed Central

    Wang, Qiang; Ruan, Xiao; Jin, Zhi-hua; Yan, Qi-chuan; Tu, Shan-jun

    2005-01-01

    An approach was established using RP-HPLC (reversed-phase high-performance liquid chromatography) to identify ten species of Rhodiola, R. coccinea A. Bor, R. junggarica C.Y. Yang et N.R. Cui spn., R. heterodonta A. Bor, R. linearifolia A. Bor, R. pamiro alaiucm A. Bor, R. kaschgarica A. Bor, R. litwinowii A. Bor, R. gelida schrenk, R. rosea L. and R. quadrifide Fisch et Mey collected from the Tianshan Mountains areas of China. Chromatograms of alcohol-soluble proteins, generated from these ten Rhodiola spp. were compared. Each chromatogram of alcohol-soluble proteins came from a single seed of one wild species only. The results showed that when using a Waters Delta Pak. C18, 5 μm particle size reversed phase column (150 mm×3.9 mm), a linear gradient of 22%–55% solvent B with a flow rate of 1 ml/min and a run time of 67 min, the chromatography gave optimum separation of Rhodiola alcohol-soluble proteins. Chromatogram of each species was different and could be used to identify those species. Cluster analysis of genetic similarity coefficients of 37% to 60% showed a medium degree of genetic diversity among the species in these eco-areas. Cluster analysis showed that the ten species of Rhodiola can be divided into four clusters and yielded the general and unique biochemical markers of these species. RP-HPLC was shown to be a rapid, repeatable and reliable method for Rhodiola species identification and analysis of genetic diversity. PMID:15909330

  9. Multiresidue HPLC methods for phenyl urea herbicides in water.

    PubMed

    Ruberu, S R; Draper, W M; Perera, S K

    2000-09-01

    High-performance liquid chromatography (HPLC) methods for the determination of phenyl urea herbicides in water are described. The target compounds include chlortoluron, diuron, fluometuron, isoproturon, linuron, metobromuron, metoxuron, monuron, neburon, and siduron. Water was subjected to solid phase extraction (SPE) using either automated SPE with 47 mm C(18) Empore disks or on-line precolumn concentration. Herbicides were separated on a C(18) reversed phase column with an acetonitile-water gradient and were detected with either a diode array detector (DAD) or a postcolumn photolysis and derivatization (PPD) detector system. Photolysis converted the phenyl ureas to monoalkylamines that were derivatized to fluorescent isoindoles by reaction with o-phthalaldehyde and 2-mercaptoethanol. The DAD monitoring at 245 nm was linear over three decades with instrument detection limits of approximately 0.01 mg/L. SPE efficiency was between 48 and 70% in laboratory reagent water, but use of the internal standard quantitation method improved accuracy. High total dissolved solids and total organic carbon values in surface water improved recoveries relative to laboratory reagent water for all of the phenyl ureas. In Colorado River water spiked at 1 or 50 microg/L, mean recoveries ranged from 74 to 104%. Method detection limits (MDLs) ranged from 4 to 40 ng/L (parts per trillion) with the DAD instrument. PPD detection was highly specific but resulted in a slight loss in chromatographic efficiency and average MDLs approximately 5 times higher using a single set of detection conditions. The study indicates that methods based on SPE followed by HPLC with diode array or PPD detection have practical utility for trace analysis of phenyl ureas in drinking water or surface waters. PMID:10995323

  10. Methods and applications of HPLC-AMS

    NASA Astrophysics Data System (ADS)

    Buchholz, Bruce A.; Dueker, Stephen R.; Lin, Yumei; Clifford, Andrew J.; Vogel, John S.

    2000-10-01

    Pharmacokinetics of physiologic doses of nutrients, pesticides, and herbicides can easily be traced in humans using a 14C-labeled compound. Basic kinetics can be monitored in blood or urine by measuring the elevation in the 14C 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. Unknowns are tentatively identified by co-elution with known standards and chemical tests that reveal functional groupings. Metabolites are quantified with the 14C 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 14C per HPLC fraction are typically achieved. Baselines are maintained by limiting injected 14C activity <0.17 Bq (4.5 pCi) on the HPLC column.

  11. 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. PMID:15158993

  12. 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

    2015-07-01

    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. PMID:26023813

  13. HPLC-fluorescence determination of individual free and conjugated bile acids in human serum.

    PubMed

    Gatti, R; Roda, A; Cerre, C; Bonazzi, D; Cavrini, V

    1997-01-01

    A method for the quantitative analysis of unconjugated and conjugated bile acids (BA) in serum of patients with primary biliary cirrhosis (PBC) before and after therapy with antibiotic or ursodeoxycholic acid (UDCA) is described. After separation of the free, glycine and taurine conjugated (F, G and T conjugated) fractions by solid-phase extraction, the isolated T conjugates were hydrolysed enzymatically using cholyglycine hydrolase. The BA fractions were derivatized using 2-bromoacetyl-6-methoxynaphthalene (Br-AMN) and detected fluorimetrically (lambda exc = 300 nm, lambda em = 460 nm). The derivatization reaction was performed under mild conditions (10 min at 40 degrees C) in an aqueous medium in the presence of tetrakis (decyl) ammonium bromide (TDeABr). The HPLC separation was achieved using an ODS column and with a mobile phase gradient mixture of A-B, where A is water and B is acetonitrile:methanol (60:40 v/v) for elution at a flow-rate of 1.2 mL/min. The reproducibility, recovery and separation of individual BA under gradient elution conditions were satisfactory, allowing a sensitive detection of each BA in serum samples with a detection limit of about 1-2 pmol. PMID:9051208

  14. 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.

  15. Impurity profiling of ibandronate sodium by HPLC-CAD.

    PubMed

    Wahl, Oliver; Holzgrabe, Ulrike

    2015-10-10

    The modern bisphosphonate drug ibandronate sodium, a challenging candidate for impurity profiling, was analyzed using high performance liquid chromatography (HPLC) combined with corona charged aerosol detection (CAD). Separation was achieved on a mixed mode column combining hydrophobic C18 and strong anion exchange retention mechanisms using a mass spectrometer compatible volatile mobile phase consisting of trifluoroacetic acid and acetonitrile while gradient elution was applied. The method was validated following the ICH guideline Q2(R1) and found suitable for the assessment of ibandronate's related substances. The observed CAD-response for all identified impurities was linear (R(2)>0.995) over a small concentration range (0.05-0.25) and a quantification limit of at least 0.03% was found. Four batches of two different manufacturers were tested by means of the method. None of the batches contained a single impurity above 0.05%. The major impurities of all batches were the synthesis by-products N-desmethyl- and N-despentyl ibandronate as well as N,N-dimethyl pamidronate. PMID:26092222

  16. HPLC retention thermodynamics of grape and wine tannins.

    PubMed

    Barak, Jennifer A; Kennedy, James A

    2013-05-01

    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. PMID:23565723

  17. Magnetic separation of algae

    DOEpatents

    Nath, Pulak; Twary, Scott N.

    2016-04-26

    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.

  18. Identification and quantification of constituents of Gardenia jasminoides Ellis (Zhizi) by HPLC-DAD-ESI-MS.

    PubMed

    Bergonzi, M C; Righeschi, C; Isacchi, B; Bilia, A R

    2012-09-15

    A simple, rapid and specific HPLC method was carried out for the analysis of characteristic constituents in Gardenia jasminoides Ellis (Zhizi), namely iridoids, caffeoyl quinic acid derivatives and crocins. The separation was successfully obtained using a C(18) column by gradient elution with mixtures of methanol and water as mobile phases; detection wavelength was set at 240 nm for iridoid glycosides, 315 nm for quinic acid derivatives and 438 nm for crocins. The analytical method was validated and the quantification of active compounds, namely iridoids, was performed. Linearity, precision, repeatability, stability, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were also reported. This assay was successfully applied for qualitative and quantitative analysis of five commercial samples of G. jasminoides Ellis. PMID:23107748

  19. HPLC method for identification and quantification of benzimidazole derivatives in antiparasitic drugs.

    PubMed

    Kulik, Anna; Białecka, Wanda; Podolska, Marzena; Kwiatkowska-Puchniarz, Barbara; Mazurek, Aleksander

    2011-01-01

    The subject of the study was to develop a versatile HPLC system for identification and determination of four benzimidazole derivatives in the antiparasitic drugs. The tests covered: Zentel, Panacur, Vermox tablets and Systamex suspension. A satisfactory separation was obtained using the Nucleosil C8 column in the gradient system composed of mobile phase A: 85% orthophosphoric acid / water / acetonitrile in 0.05:75:25, v/v/v ratio, and mobile phase B: 85% orthophosphoric acid / water / acetonitrile in 0.05:50:50, v/v/v ratio. Both phases were adjusted to pH = 4.5 with 15% sodium hydroxide solution. A detection at 288 nm for oxfendazole and 254 nm for albendazole, fenbendazole and mebendazole was applied. The correlation coefficients in the range 0,9997 - 0,9999 proved that the calibration curves were linear. The method was validated in terms of selectivity, accuracy and precision. PMID:22125945

  20. 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. PMID:26660172

  1. Multiecho IDEAL Gradient-Echo Water-Fat Separation for Rapid Assessment of Cartilage Volume at 1.5 T: Initial Experience1

    PubMed Central

    Chen, Christina A.; Lu, Wenmiao; John, Chand T.; Hargreaves, Brian A.; Reeder, Scott B.; Delp, Scott L.; Siston, Robert A.; Gold, Garry E.

    2009-01-01

    Institutional review board approval and informed consent were obtained for this HIPAA-compliant study. The purpose was to prospectively compare multiecho iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) gradient-echo (GRE) magnetic resonance (MR) imaging with three-dimensional fat-suppressed (FS) spoiled GRE (SPGR) MR imaging to evaluate the articular cartilage of the knee. Six healthy volunteer and 10 cadaver knees were imaged at 1.5 T. Signal-to-noise ratio (SNR), SNR efficiency, and cartilage volume were measured. SNR and SNR efficiency were significantly higher with multiecho IDEAL GRE than with FS SPGR imaging (P < .031). Both methods produced equivalent cartilage volumes (overall concordance correlation coefficient, 0.998) with high precision and accuracy. The use of a cartilage phantom confirmed high accuracy in volume measurements and high reproducibility for both methods. Multiecho IDEAL GRE provides high signal intensity in cartilage and synovial fluid and is a promising technique for imaging articular cartilage of the knee. PMID:19528355

  2. 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. PMID:18446772

  3. 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. PMID:24552644

  4. 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)

  5. 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

  6. A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells

    PubMed Central

    Appala, Raju N.; Appala, Raju V. V. S. S.

    2016-01-01

    A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cells. Glutathione is a major intracellular antioxidant having multiple biological effects, best known for its cytoprotective effects against cell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis. Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection. The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm. The developed HPLC-UV method was validated with respect to precision, accuracy, robustness, and linearity within a range of 1–20 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively. Furthermore, the method shows the applicability for monitoring the oxidative stress in PC-12 cells. PMID:27127683

  7. HPLC method development, validation, and impurity characterization of a potent antitumor indenoisoquinoline, LMP776 (NSC 725776).

    PubMed

    Wang, Jennie; Liu, Mingtao; Yang, Chun; Wu, Xiaogang; Wang, Euphemia; Liu, Paul

    2016-05-30

    An HPLC method for the assay of a DNA topoisomerase inhibitor, LMP776 (NSC 725776), has been developed and validated. The stress testing of LMP776 was carried out in accordance with International Conference on Harmonization (ICH) guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of LMP776 from its impurities and degradation products was achieved within 40 min on a Supelco Discovery HS F5 column (150 mm × 4.6 mm i.d., 5 μm) with a gradient mobile phase comprising 38-80% acetonitrile in water, with 0.1% trifluoroacetic acid in both phases. LC/MS was used to obtain mass data for characterization of impurities and degradation products. One major impurity was isolated through chloroform extraction and identified by NMR. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25-0.75 mg/mL, r = 0.9999), accuracy (recovery 98.6-100.4%), precision (RSD ≤ 1.4%), and sensitivity (LOD 0.13 μg/mL). The validated method was used in the stability study of the LMP776 drug substance in conformance with the ICH Q1A (R2) guideline. PMID:26970596

  8. HPLC analysis of isoflavonoids and other phenolic agents from foods and from human fluids.

    PubMed

    Franke, A A; Custer, L J; Wang, W; Shi, C Y

    1998-03-01

    A fast, precise and selective diode array HPLC method is presented for the extraction and analysis of soy isoflavonoids from foods and from human urine, plasma, and breast milk in support of mechanistic and epidemiologic studies assessing the potential cancer protective role of soya or isoflavones. Solid phase or solvent extraction was chosen for isolation, and enzymatic or acid hydrolysis procedures were used for aglycone production depending on the matrix to be analyzed. C-18 reversed-phase HPLC was applied to selectively separate and quantitate daidzein (1), glycitein (3), and genistein (4), including their malonyl (a) and acetyl (b) esters, and their mammalian metabolites equol (6) and O-desmethylangolensin (7), as well as formononetin (2), biochanin-A (5), and coumestrol (8) using a gradient elution system. UV absorbance scans and authentic standards were applied for identification purposes, additional to fluorometric monitoring, electrochemical detection, and GC/ MS analysis after trimethyl silylation. Detection limits of 20-microl injections were found to be 1.09, 0.53, 3.28, and 1.00 pmoles for daidzein, genistein, equol, and O-desmethylangolensin (DMA), respectively, by monitoring at the individual compound's absorption maximum. The proposed method was applied to monitor isoflavone levels in soy foods and in human plasma, urine and breast milk after challenge with roasted soybeans. Implications of the presented results on the potential activity of isoflavones to prevent cancer by exposing newborn infants to these agents are discussed. PMID:9492334

  9. 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%. PMID:25713108

  10. Reversed phase-HPLC for rapid determination of polyphenols in flowers of rose species.

    PubMed

    Kumar, Neeraj; Bhandari, Pamita; Singh, Bikram; Gupta, Ajai P; Kaul, Vijay K

    2008-02-01

    A rapid, simple, sensitive, robust, and improved HPLC method was developed and validated for determination of 10 polyphenols, namely gallic acid, catechin, epicatechin, rutin, m-coumaric acid, quercitrin, myricetin, quercetin, apigenin, and kaempferol in fresh flowers of Rosa bourboniana and R. brunonii and in both fresh flowers and marc (left after industrial distillation of rose oil) of R. damascena. Six polyphenols, gallic acid, rutin, quercitrin, myricetin, quercetin, and kaempferol, were detected and quantified in all extracts. The chromatographic separation of 10 polyphenols was achieved in less than 16 min by RP-HPLC (Phenomenex, Luna C18 (2) column, 5 microm, 250 mm x 4.6 mm) using linear gradient elution of water and acetonitrile (0.02% trifluroacetic acid) with a flow rate of 1 mL/min at lambda 280 nm. Standard calibration curves were linear in the range of 0.39-500 microg/mL. Good results were achieved with respect to repeatability (RSD <3%) and recovery (98.6-100.8%). The method was validated for linearity, accuracy, repeatability, LOD, and LOQ. PMID:18172921