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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. 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…

  3. 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…

  4. 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 ...

  5. Partial separation of fullerenes by gradient sublimation

    SciTech Connect

    Yeretzian, C.; Wiley, J.B.; Holczer, K.; Su, T.; Nguyen, S.; Kaner, R.B.; Whetten, R.L. )

    1993-09-30

    An experimental technique is investigated to separate/enrich fullerenes of metallofullerenes, exploiting differences in sublimation temperatures without the use of solvents. Fullerenes are sublimed out of the soot and deposited on a quartz rod along a temperature gradient (gradient sublimation). In a position-sensitive experiment the composition of the deposit on the rod is monitored by laser-desorption mass spectrometry. Strongly enriched regions containing specific fullerene molecules (i.e., C[sub 84] or LaC[sub 82]) are observed. Furthermore, C[sub 74], which could not be extracted from the soot by organic solvents, sublimes out of the soot. 26 refs., 6 figs.

  6. Dynamic separation of macromolecules under temperature gradient

    NASA Astrophysics Data System (ADS)

    Maeda, Yusuke; Buguin, Axel; Libchaber, Albert

    2011-03-01

    Thermophoresis is a motion of suspensions in a fluid that are subjected to a temperature gradient. Although its effect is widely studied in case of single solute in water, little is known about how the mixture of different solutes is affected. We heated water with an infrared laser by ΔTmax = 5C and ∇ T = 0.25C/um to induce thermophoresis of polyethylene glycol (PEG) and DNA. PEG is depleted from the hot region and results in a stationary gradient of its high volume fraction ϕ . Under this high concentration of PEG, DNA of small concentration is submitted to thermophoresis and osmotic pressure difference. The DNA shows regime of depletion, ring-like localization and accumulation as the volume fraction of PEG increases. As the osmotic force depends on the size of trapped solutes, DNA of different size accumulates at different regions. Depending whether the DNA size is below or above 5kbp a different scaling of position versus DNA size is observed. Thermal separation is a general phenomenon. It applies also to RNA and microbeads. YTM is supported by JSPS fellowship and M.Josee-H.Kravis fellowship from the Rockefeller University.

  7. Separation of {sup 32}P-postlabeled DNA adducts of polycyclic aromatic hydrocarbons and nitrated polycyclic aromatic hydrocarbons by HPLC

    SciTech Connect

    King, L.C.; Gallagher, J.E.; Lewtas, J.; George, M.

    1994-07-01

    The {sup 32}P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO{sub 2}-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO{sub 2}-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO{sub 2}-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. A second NO{sub 2}-PAH HPLC gradient system was developed to separate NO{sub 2}-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO{sub 2}-PAH-DNA adducts were compared using both adduct enhancement versions of the {sup 32}P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO{sub 2}-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the {sup 32}P-postlabeled DNA adduct standards (PAHs and NO{sub 2}-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO{sub 2}-PAH standards used in this study. HPLC analyses of the NO{sub 2}-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.

  8. 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.

  9. PLETHORA gradient formation mechanism separates auxin responses

    PubMed Central

    Siligato, Riccardo; Smetana, Ondřej; Díaz-Triviño, Sara; Salojärvi, Jarkko; Wachsman, Guy; Prasad, Kalika; Heidstra, Renze; Scheres, Ben

    2015-01-01

    During plant growth, dividing cells in meristems must coordinate transitions from division to expansion and differentiation, thus generating three distinct developmental zones: the meristem, elongation zone and differentiation zone1. Simultaneously, plants display tropisms, rapid adjustments of their direction of growth to adapt to environmental conditions. It is unclear how stable zonation is maintained during transient adjustments in growth direction. In Arabidopsis roots, many aspects of zonation are controlled by the phytohormone auxin and auxin-induced PLETHORA (PLT) transcription factors, both of which display a graded distribution with a maximum near the root tip2-12. In addition, auxin is also pivotal for tropic responses13,14. Here, using an iterative experimental and computational approach, we show how an interplay between auxin and PLTs controls zonation and gravitropism. We find that the PLT gradient is not a direct, proportionate readout of the auxin gradient. Rather, prolonged high auxin levels generate a narrow PLT transcription domain from which a gradient of PLT protein is subsequently generated through slow growth dilution and cell-to-cell movement. The resulting PLT levels define the location of developmental zones. In addition to slowly promoting PLT transcription, auxin also rapidly influences division, expansion and differentiation rates. We demonstrate how this specific regulatory design in which auxin cooperates with PLTs through different mechanisms and on different timescales enables both the fast tropic environmental responses and stable zonation dynamics necessary for coordinated cell differentiation. PMID:25156253

  10. 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.

  11. 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

  12. 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.

  13. HPLC SEPARATION OF CHIRAL ORGANOPHOSPHORUS PESTICIDES ON POLYSACCHARIDE CHIRAL STATIONARY PHASES

    EPA Science Inventory

    High-performance liquid chromatographic separation of the individual enantiomers of 12 organophosphorus pesticides (OPs) were obtained on polysaccharide chiral HPLC columns using an alkane-alcohol mobile phase. The OP pesticides were crotoxyphos, dialifor, dyfonate, fenamiphos, ...

  14. 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.

  15. Temperature Gradients Induce Phase Separation in a Miscible Polymer Solution

    NASA Astrophysics Data System (ADS)

    Kumaki, Jiro; Hashimoto, Takeji; Granick, Steve

    1996-09-01

    Phase separation occurred up to 20 °C above the coexistence temperature in a polymer solution (polystyrene-polybutadiene-dioctylphthalate) to which small temperature gradients ( ~2 °C) were applied. Before convection began, spinodal-like patterns with characteristic spacing that grew in proportion to time elapsed persisted for times up to hours. The cause appears to be thermally driven concentration gradients normal to the surface, large enough to induce phase separation parallel to the surface, although temperatures throughout the mixture exceeded the thermodynamic coexistence temperature.

  16. 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

  17. 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.

  18. 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...

  19. Adaptive improved natural gradient algorithm for blind source separation.

    PubMed

    Liu, Jian-Qiang; Feng, Da-Zheng; Zhang, Wei-Wei

    2009-03-01

    We propose an adaptive improved natural gradient algorithm for blind separation of independent sources. First, inspired by the well-known backpropagation algorithm, we incorporate a momentum term into the natural gradient learning process to accelerate the convergence rate and improve the stability. Then an estimation function for the adaptation of the separation model is obtained to adaptively control a step-size parameter and a momentum factor. The proposed natural gradient algorithm with variable step-size parameter and variable momentum factor is therefore particularly well suited to blind source separation in a time-varying environment, such as an abruptly changing mixing matrix or signal power. The expected improvement in the convergence speed, stability, and tracking ability of the proposed algorithm is demonstrated by extensive simulation results in both time-invariant and time-varying environments. The ability of the proposed algorithm to separate extremely weak or badly scaled sources is also verified. In addition, simulation results show that the proposed algorithm is suitable for separating mixtures of many sources (e.g., the number of sources is 10) in the complete case.

  20. 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...

  1. 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.

  2. Magnet design for superconducting open gradient magnetic separator

    NASA Astrophysics Data System (ADS)

    Ahoranta, Maria; Lehtonen, Jorma; Mikkonen, Risto

    2003-04-01

    The use of superconductivity opens new applications for magnetic separation because very high magnetic fields become available. In this paper the magnet design for a laboratory scale superconducting open gradient magnetic separator is presented. The separator will be used to optimize the separation parameters for different kinds of applications, such as the foundry sand purification. Therefore, the goal of the magnet design is to obtain a constant magnetic force density distribution inside the working volume. The high magnitude of magnetic force density is required because the materials to be separated have low magnetic susceptibilities. The maximum achievable force density is determined by the critical current in superconducting magnets. The advantages and drawbacks of solenoid, racetrack and saddle coil geometries are compared. Ways for improving the performance of the system is discussed. Finally, the influence of the stray field on the slurry flow outside the working volume is studied.

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

    DOE PAGES

    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

  4. 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

  5. [Consideration on high gradient magnetic separation of red cells].

    PubMed

    Iacob, Gh; Ciochină, Al D; Herea, D D; Dimitriu, Cristina; Chiruţă, Roxana; Vieru, Cristina; Stratone, Ana

    2004-01-01

    The red cells exhibit a proper magnetism due to hemoglobin. In a prototype of high gradient magnetic separator equipped with an ordered ferromagnetic matrix, a set of experiments with blood to determine the influence of the process parameters on the efficiency of the erythrocytes capture was realized. Dependent on the values of the magnetic induction (1.76-2.01 T), average blood flow velocity through the matrix (0.55-1.1 mm/s), and stationary time in the matrix, different values of concentration for the red cells in the matrix were obtained. For tests realized with integral blood, the concentration was approximately 20%, while for tests with diluted blood the concentration fluctuated from 14.51% to 29.90%. A blood recirculation through the matrix led to a concentration of 37.86%. The magnetic separation method permits an acceptable red cells concentration, without apparent destructive effects on the cells.

  6. Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC.

    PubMed

    Bonfatti, Valentina; Giantin, Mery; Rostellato, Roberta; Dacasto, Mauro; Carnier, Paolo

    2013-01-15

    A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC.

  7. Blind separation of positive sources by globally convergent gradient search.

    PubMed

    Oja, Erkki; Plumbley, Mark

    2004-09-01

    The instantaneous noise-free linear mixing model in independent component analysis is largely a solved problem under the usual assumption of independent nongaussian sources and full column rank mixing matrix. However, with some prior information on the sources, like positivity, new analysis and perhaps simplified solution methods may yet become possible. In this letter, we consider the task of independent component analysis when the independent sources are known to be nonnegative and well grounded, which means that they have a nonzero pdf in the region of zero. It can be shown that in this case, the solution method is basically very simple: an orthogonal rotation of the whitened observation vector into nonnegative outputs will give a positive permutation of the original sources. We propose a cost function whose minimum coincides with nonnegativity and derive the gradient algorithm under the whitening constraint, under which the separating matrix is orthogonal. We further prove that in the Stiefel manifold of orthogonal matrices, the cost function is a Lyapunov function for the matrix gradient flow, implying global convergence. Thus, this algorithm is guaranteed to find the nonnegative well-grounded independent sources. The analysis is complemented by a numerical simulation, which illustrates the algorithm. PMID:15265323

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

    PubMed

    Chandra, A; Rana, J; Li, Y

    2001-08-01

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

  9. Development of a Gradient HPLC Method for the Simultaneous Determination of Sotalol and Sorbate in Oral Liquid Preparations Using Solid Core Stationary Phase

    PubMed Central

    2015-01-01

    A selective and sensitive gradient HPLC-UV method for quantification of sotalol hydrochloride and potassium sorbate in five types of oral liquid preparations was developed and fully validated. The separation of an active substance sotalol hydrochloride, potassium sorbate (antimicrobial agent), and other substances (for taste and smell correction, etc.) was performed using an Ascentis Express C18 (100 × 4.6 mm, particles 2.7 μm) solid core HPLC column. Linear gradient elution mode with a flow rate of 1.3 mL min−1 was used, and the injection volume was 5 µL. The UV/Vis absorbance detector was set to a wavelength of 237 nm, and the column oven was conditioned at 25°C. A sodium dihydrogen phosphate dihydrate solution (pH 2.5; 17.7 mM) was used as the mobile phase buffer. The total analysis time was 4.5 min (+2.5 min for reequilibration). The method was successfully employed in a stability evaluation of the developed formulations, which are now already being used in the therapy of arrhythmias in pediatric patients; the method is also suitable for general quality control, that is, not only just for extemporaneous preparations containing the mentioned substances. PMID:25878920

  10. Development of a gradient HPLC method for the simultaneous determination of sotalol and sorbate in oral liquid preparations using solid core stationary phase.

    PubMed

    Matysova, Ludmila; Zahalkova, Oxana; Klovrzova, Sylva; Sklubalova, Zdenka; Solich, Petr; Zahalka, Lukas

    2015-01-01

    A selective and sensitive gradient HPLC-UV method for quantification of sotalol hydrochloride and potassium sorbate in five types of oral liquid preparations was developed and fully validated. The separation of an active substance sotalol hydrochloride, potassium sorbate (antimicrobial agent), and other substances (for taste and smell correction, etc.) was performed using an Ascentis Express C18 (100 × 4.6 mm, particles 2.7 μm) solid core HPLC column. Linear gradient elution mode with a flow rate of 1.3 mL min(-1) was used, and the injection volume was 5 µL. The UV/Vis absorbance detector was set to a wavelength of 237 nm, and the column oven was conditioned at 25°C. A sodium dihydrogen phosphate dihydrate solution (pH 2.5; 17.7 mM) was used as the mobile phase buffer. The total analysis time was 4.5 min (+2.5 min for reequilibration). The method was successfully employed in a stability evaluation of the developed formulations, which are now already being used in the therapy of arrhythmias in pediatric patients; the method is also suitable for general quality control, that is, not only just for extemporaneous preparations containing the mentioned substances. PMID:25878920

  11. Medical protein separation system using high gradient magnetic separation by superconducting magnet

    NASA Astrophysics Data System (ADS)

    Kamioka, Y.; Agatsuma, K.; Kajikawa, K.; Ueda, H.; Furuse, M.; Fuchino, S.; Iitsuka, T.; Nakamura, S.

    2014-01-01

    A high gradient magnetic separation system for medical protein using affinity magnetic nano-beads has been developed. Medical protein such as monoclonal antibody or immunoglobulin is an important substance as a medicine for cancer etc. However; the separation system of these medical protein has very low separation rate and the cost of product is extremely high. The developed system shows very high separation efficiency and can achieve low cost by large production rate compared to the system now using in this field. The system consists of a 3T superconducting magnet cooled by a cryo-cooler, a filter made of fine magnetic metal wires of about 30μm diameter and a demagnetization circuit and a liquid circulation pump for solvent containing medical protein. Affinity magnetic nano-beads is covered with the medical protein after agitation of solvent containing the protein and nano-beads, then the solvent flows through the system and the beads are trapped in the filters by high gradient magnetic field. The beads are released and flow out of the system by the AC demagnetization of the filters using LC resonance circuits after discharge of the magnet. The test results shows 97.8% of the magnetic nano-beads in pure water were captured and 94.1% of total beads were collected.

  12. Simultaneous separation and determination of process-related substances and degradation products of venlafaxine by reversed-phase HPLC.

    PubMed

    Nageswara Rao, R; Narasa Raju, A

    2006-12-01

    A simple and rapid gradient RP HPLC method for simultaneous separation and determination of venlafaxine and its related substances in bulk drugs and pharmaceutical formulations has been developed. As many as four process impurities and one degradation product of venlafaxine have been separated on a Kromasil KR100-5C18 (4.6 mm x 250 mm; particle size 5 microm) column with gradient elution using 0.3% diethylamine buffer (pH 3.0) and ACN/methanol (90:10 v/v) as a mobile phase. The column was maintained at 40 degrees C and the eluents were monitored with photo diode array detection at 225 nm. The chromatographic behaviour of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. The inter- and intraday assay precision was < 4.02% (%RSD) and the recoveries were in the range of 96.19-101.14% with %RSD < 1.15%. The correlation coefficients (r2) for calibration curves of venlafaxine as well as impurities were in the range of 0.9942-0.9999. The proposed RP-LC method was successfully applied to the analysis of commercial formulations and the recoveries of venlafaxine were in the range of 99.32-100.67 with %RSD <0.58%. The method could be of use not only for rapid and routine evaluation of the quality of venlafaxine in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Forced degradation of venlafaxine was carried out under thermal, photo, acidic, basic and peroxide conditions and the acid degradation products were characterized by ESI-MS/MS, 1H NMR and FT-IR spectral data.

  13. Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC.

    PubMed

    Bonfatti, Valentina; Giantin, Mery; Rostellato, Roberta; Dacasto, Mauro; Carnier, Paolo

    2013-01-15

    A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC. PMID:23122071

  14. Application of statistical design for the optimization of amino acid separation by reverse-phase HPLC.

    PubMed

    Gheshlaghi, R; Scharer, J M; Moo-Young, M; Douglas, P L

    2008-12-01

    Modified resolution and overall separation factors used to quantify the separation of complex chromatography systems are described. These factors were proven to be applicable to the optimization of amino acid resolution in reverse-phase (RP) HPLC chromatograms. To optimize precolumn derivatization with phenylisothiocyanate, a 2(5-1) fractional factorial design in triplicate was employed. The five independent variables for optimizing the overall separation factor were triethylamine content of the aqueous buffer, pH of the aqueous buffer, separation temperature, methanol/acetonitrile concentration ratio in the organic eluant, and mobile phase flow rate. Of these, triethylamine concentration and methanol/acetonitrile concentration ratio were the most important. The methodology captured the interaction between variables. Temperature appeared in the interaction terms; consequently, it was included in the hierarchic model. The preliminary model based on the factorial experiments was not able to explain the response curvature in the design space; therefore, a central composite design was used to provide a quadratic model. Constrained nonlinear programming was used for optimization purposes. The quadratic model predicted the optimal levels of the variables. In this study, the best levels of the five independent variables that provide the maximum modified resolution for each pair of consecutive amino acids appearing in the chromatograph were determined. These results are of utmost importance for accurate analysis of a subset of amino acids.

  15. 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.

  16. 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

  17. Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives.

    PubMed Central

    Spivak, W; Carey, M C

    1985-01-01

    We describe a facile and sensitive reverse-phase h.p.l.c. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. The method can be 'scaled up' for preparative isolation of pure BDG and BMG from pigment-enriched biles. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO . UCB) was employed as a single h.p.l.c. reference standard for quantification of BMG and BDG. We demonstrate that: separation and quantification of biliary bile pigments are rapid (approximately 25 min); bile pigment concentrations ranging from 1-500 microM can be determined 'on line' by using 5 microliters of bile without sample pretreatment; bilirubin conjugates can be obtained preparatively in milligram quantities without degradation or contamination by other components of bile. H.p.l.c. analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1 to 17 microM. These values are considerably lower than those estimated previously by t.l.c. BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster

  18. 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.

  19. Optimization of separation and determination of moxifloxacin and its related substances by RP-HPLC.

    PubMed

    Djurdjevic, Predrag; Ciric, Andrija; Djurdjevic, Aleksandra; Stankov, Milena Jelikic

    2009-09-01

    A RP-HPLC method for the separation and determination of impurities of moxifloxacin, in its pharmaceutical forms as well as moxifloxacin degradation products, was developed with the aid of DryLab software and chemometric (response surface) approach. The separation of four synthesis-related impurities was achieved on a Waters C(18) XTerra column using a mobile phase of (water+triethylamine (2%, v/v)): acetonitrile=90:10 (v/v%); the pH of water phase being adjusted with phosphoric acid to 6.0. Flow rate of the mobile phase was 1.5 ml/min and UV detection at 290 nm was employed. The column was thermostated at 45 degrees C. The resolution between the two least resolved impurity peaks was in average, R(s,min) > 1.5. Method validation parameters indicate linear dynamic range 0.2-2.0 microg/ml with LOQ ca. 0.20 microg/ml and LOD ca. 0.05 microg/ml for all analytes. The method was applied for the impurities determination in drug tablets and infusion (Avelox), Bayer AG) and for degradation products determination in a stability study of moxifloxacin. The impurity content in the tablets and infusion was quantified as 0.1% of total drug. Two degradation products were noted under hydrolytic conditions. The method can also be used for rapid and accurate quantification of moxifloxacin hydrochloride in its tablets during stability testing.

  20. 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

  1. 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.

  2. 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

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

    PubMed

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

    2005-01-01

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

  4. Real-time separation of natural products by ultrafast 2D NMR coupled to on-line HPLC.

    PubMed

    Queiroz, Luiz H K; Queiroz, Darlene P K; Dhooghe, Liene; Ferreira, Antonio G; Giraudeau, Patrick

    2012-05-21

    Hyphenated HPLC-NMR is an extremely efficient analytical tool, which makes it possible to perform on-flow experiments where 1D NMR spectra are obtained in real time as the analytes are separated and eluted from the chromatographic column. However, it is incompatible with multidimensional NMR methods that form an indispensible tool for the study of complex mixtures. Recently, Frydman and co-workers have proposed an ultrafast 2D NMR approach, where a complete 2D NMR correlation can be recorded in a single scan, thus providing a solution to the irreversibility of hyphenated techniques. This paper presents the first implementation of on-line ultrafast HPLC-NMR. Ultrafast COSY spectra are acquired every 12 s in the course of a chromatographic run performed on a mixture of natural aromatic compounds. The results, obtained on a commercial HPLC-NMR setup, highlight the generality of the ultrafast HPLC-NMR methodology, thus opening the way to a number of applications in the numerous fields in which HPLC-NMR forms a routine analytical tool.

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

    PubMed

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

    2013-11-15

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

  6. 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

  7. '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.

  8. Peak capacity optimization of peptide separations in reversed-phase gradient elution chromatography: fixed column format.

    PubMed

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

    2006-05-15

    The optimization of peak capacity in gradient elution RPLC is essential for the separation of multicomponent 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 (phi(final)) 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 phi(final). 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.

  9. Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation.

    PubMed

    Jackson, C; Dench, J E; Hall, D O; Moore, A L

    1979-07-01

    Discontinuous Percoll density gradients have been developed for the purification of mitochondria, permitting rapid separation under isosmotic and low viscosity conditions. Mitochondria from several etiolated tissues have been successfully separated from contaminating subcellular structures by this method. For potato tuber the ratio of washed to purified mitochondrial protein was 2.6, similar to the increase in specific activity of cytochrome c oxidase following separation. The purification of mitochondria from green leaf tissues on Percoll gradients has reduced chlorophyll contamination of spinach mitochondria from about 70 micrograms chlorophyll per milligram protein to approximately 8 micrograms chlorophyll per milligram protein.The ratio of protein content of the washed mitochondria compared to that in the purified preparation was 7 for spinach and respiratory activity was retained. The physiological integrity and oxidative properties of washed and gradient mitochondria are compared. PMID:16660904

  10. 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.

  11. 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

  12. 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

  13. Separation of cardiac myosin heavy chains by gradient SDS-PAGE.

    PubMed

    Esser, K A; Boluyt, M O; White, T P

    1988-09-01

    Separation of alpha- and beta-myosin heavy chains (MHCs) in cardiac ventricles of rats by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was accomplished and compared with the separation of myosin isozymes obtained with pyrophosphate gels. Whole muscle homogenates were electrophoresed on a 4-9% linear gradient SDS polyacrylamide gel for 3-4 h. MHC bands were identified by the migration distance relative to a MHC standard and immunoblot results with a monoclonal antibody to MHC. The MHC bands were further identified as alpha and beta based on the electrophoretic mobility of ventricular homogenates from hypothyroid and hyperthyroid rats and ventricular and slow soleus skeletal muscle homogenates from control rats. The beta-MHC migrated faster than alpha-MHC, and laser densitometry revealed separate peaks when both MHCs were present. With homogenates containing MHC ranging from 0 to 100% alpha, the separation of MHCs with gradient SDS-PAGE correlated highly (r = 0.97) with separation of myosin isozymes by pyrophosphate gel electrophoresis. The SDS-PAGE technique reported herein is a quick, valid, and direct method for the identification and quantification of ventricular MHCs.

  14. Formation of metastable structures by phase separation triggered by initial composition gradients in thin films.

    PubMed

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

    2012-08-14

    Phase separation kinetics of a binary (A,B) mixture contained in a thin film of thickness D induced by a quench from the one-phase region into the miscibility gap is studied by simulations using a Cahn-Hilliard-Cook model. The initial randomly mixed state (50% A, 50% B) contains a concentration gradient perpendicular to the film, while the surfaces of the film are "neutral" (no preference for either A or B). In thermal equilibrium, a pattern of large A-rich and B-rich domains must result, separated by domain walls oriented perpendicularly to the external surfaces of the thin film. However, it is shown that for many choices of D and the strength of the initial gradient Ψ(g), instead a very long-lived metastable layered structure forms, with two domains separated by a single interface parallel to the external walls. The transient time evolution that leads to this structure is interpreted in terms of a competition between domain growth in the bulk and surface-directed spinodal decomposition caused by the gradient during the initial stages. A surprising and potentially useful finding is that a moderate concentration gradient perpendicular to the film does not favor the layered structure but facilitates the approach toward the true equilibrium with just two domain walls perpendicular to the film. This mechanism may have useful applications in producing layered materials. PMID:22897299

  15. Simple automated liquid chromatographic system for splitless nano column gradient separations.

    PubMed

    Sesták, Jozef; Duša, Filip; Moravcová, Dana; Kahle, Vladislav

    2013-02-01

    A simple splitless gradient liquid chromatographic system for micro and nano column separations has been assembled and tested. It consists of an OEM programmable syringe pump equipped with a glass microsyringe and ten-port selector valve. Gradient of mobile phase was created in the syringe barrel due to turbulent mixing. Capability of suggested system to create various gradient profiles was verified using 50-μl, 100-μl, and 250-μl glass syringes. Acetone, thiourea, and uracil were tested as gradient markers and finally, uracil was proved to be an excellent way of water-acetonitrile gradient tracing. It was found that up to 80% of the total syringe volume is available as a linear gradient section. In context to micro and nano column chromatography, the best results were obtained using the 100-μl syringe. Separations were performed on the capillary monolithic column Chromolith CapRod RP-18e (150mm×0.1mm) and system performance was evaluated using a test mixture of six alkylphenones as well as tryptic digest of bovine serum albumin. Results proved that suggested system is able to create uniform gradients with high repeatability of retention times of test solutes (RSD<0.3%). Repeatability of injection of sample volumes in the range of 0.1-3μl was evaluated using on-column preconcentration technique which means that sample was diluted in mobile phase of low eluting strength. Repeatability of the peak areas was measured and statistically evaluated (RSD<5%).

  16. 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.

  17. 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.

  18. Magnetic separation of FCC equilibrium catalyst by HGMS. [Fluid catalytic cracking (FCC); high gradient magnetic separator (HGMS)

    SciTech Connect

    Takase, S.; Ushio, M.; Oishi, Y.; Morita, T.; Shiori, T.

    1982-09-01

    The results of a study on the magnetic separation of a fluid catalytic cracking catalysts by a high gradient magnetic separator are presented. Seven nickel-impregnated catalysts (Ni content ranged from 0.0 to 4.45 wt.%) were mixed in equal amounts as a model sample. The velocity effect of carrier gas is detailed with the magnetic yield and magnetic susceptibility plotted against magnetic field at a constant carrier gas velocity. The distribution of magnetic susceptibility of separated components was also studied. The results of the study suggested that highly metal-contaminated catalysts can be selectively separated by HGMS according to metal content. Actual equilibrium catalysts used with heavy feedstocks are also expected to separate into mags and nonmags, and in such real cases, nonmags have larger surface areas and higher catalytic activity as well as selectivity. As such, it was concluded that a magnetic separation of highly metal-contaminated catalysts by HGMS followed by the make-up of fresh catalyst gives rise to a considerable reduction of catalyst consumption in residual oil FCC processing. (JMT)

  19. 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

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

    PubMed

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

    2000-06-01

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

  1. Isolation of mono-caffeoylquinic acids from tobacco waste using continuous resin-based pre-separation and preparative HPLC.

    PubMed

    Wang, Jun; Lu, Dingqiang; Liang, Yao; Zhao, Hui; Luo, Min; Ling, Xiuquan; Ouyang, Pingkai

    2012-06-01

    Three isomers of mono-caffeoylquinic acid, specifically, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid, were successfully isolated from a crude extract of tobacco (Nicotiana tobaccum L.) wastes using continuous resin-based pre-separation and preparative high-performance liquid chromatography (HPLC). The extract of tobacco wastes was continuously pre-separated by resin-based columns packed with D101 and XAD-4, yielding total mono-caffeoylquinic acids with a purity of 67.71% and a recovery rate of 90.06%. Variables affecting resolution and productivity of three mono-caffeoylquinic acid isomers in preparative HPLC (i.e. mobile-phase composition, pH, flow rate and loading amount) were studied. The optimum chromatographic conditions were determined to be a mobile phase consisting of 15% (v/v) methanol and aqueous acetic acid with a pH of 4.5, a flow rate of 4.0 mL/min, a loading amount of 4 mL and a detection wavelength of 360 nm. From 300 mg of loading sample, 56.3 mg of 3-O-caffeoylquinic acid, 92.8 mg of 5-O-caffeoylquinic acid and 73.1 mg of 4-O-caffeoylquinic acid were obtained in a single run, each with a purity of over 98% by HPLC. The structures of the isolated compounds were elucidated by ESI-MS, (1) H-NMR and (13) C-NMR spectral data.

  2. 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.

  3. 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.

  4. 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

  5. Reverse-phase HPLC separation of hemp seed (Cannabis sativa L.) protein hydrolysate produced peptide fractions with enhanced antioxidant capacity.

    PubMed

    Girgih, Abraham T; Udenigwe, Chibuike C; Aluko, Rotimi E

    2013-03-01

    Hemp seed protein hydrolysate (HPH) was produced through simulated gastrointestinal tract (GIT) digestion of hemp seed protein isolate followed by partial purification and separation into eight peptide fractions by reverse-phase (RP)-HPLC. The peptide fractions exhibited higher oxygen radical absorbance capacity as well as scavenging of 2,2-diphenyl-1-picrylhydrazyl, superoxide and hydroxyl radicals when compared to HPH. Radical scavenging activities of the fractionated peptides increased as content of hydrophobic amino acids or elution time was increased, with the exception of hydroxyl radical scavenging that showed decreased trend. Glutathione (GSH), HPH and the RP-HPLC peptide fractions possessed low ferric ion reducing ability but all had strong (>60 %) metal chelating activities. Inhibition of linoleic acid oxidation by some of the HPH peptide fractions was higher at 1 mg/ml when compared to that observed at 0.1 mg/ml peptide concentration. Peptide separation resulted in higher concentration of some hydrophobic amino acids (especially proline, leucine and isoleucine) in the fractions (mainly F5 and F8) when compared to HPH. The elution time-dependent increased concentrations of the hydrophobic amino acids coupled with decreased levels of positively charged amino acids may have been responsible for the significantly higher (p < 0.05) antioxidant properties observed for some of the peptide fractions when compared to the unfractionated HPH. In conclusion, the antioxidant activity of HPH after simulated GIT digestion is mainly influenced by the amino acid composition of some of its peptides.

  6. 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.

  7. Screening Approach for Chiral Separation of β-Aminoketones by HPLC on Various Polysaccharide-Based Chiral Stationary Phases.

    PubMed

    Addadi, Khadidja; Sekkoum, Khaled; Belboukhari, Nasser; Cheriti, Abdelkrim; Aboul-Enein, Hassan Y

    2015-05-01

    Nine β-aminoketones were synthesized via Mannich reaction when benzaldehyde was condensed with some primary amines and acetophenone. The purified compounds were identified by using spectroscopic methods. The enantiomeric separation of these derivatives was carried out by high-performance liquid chromatography (HPLC) using several coated and immobilized polysaccharide stationary phases, namely, Chiralcel(®) OD-H, Chiralcel(®) OD, Chiralcel(®) OJ, Chiralpak(®) AD, Chiralpak(®) IA, and Chiralpak(®) IB using different mobile phases composed of n-hexane and alcohol mixed in various ratios or pure ethanol or isopropanol. The retention behavior and selectivity of these chiral stationary phases were examined in isocratic normal phase mode. The results indicate that cellulose derivatives have higher enantioselectivity than amylose derivatives for the separation of racemic β-amino ketones. PMID:25752940

  8. 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.

  9. 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.

  10. Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides.

    PubMed

    Park, Sung-Soo; Maudsley, Stuart

    2011-02-01

    Global profiling of phosphoproteomes has proven to be 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 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 titanium dioxide chromatography. Using this process, we 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 pHs 9.2 and 9.4. High-resolution separation of multiply phosphorylated peptides was primarily achieved using elution ranges greater than 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.

  11. 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

  12. 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.

  13. Simultaneous separation and determination of fructose, sorbitol, glucose and sucrose in fruits by HPLC-ELSD.

    PubMed

    Ma, Chunmei; Sun, Zhen; Chen, Changbao; Zhang, Lili; Zhu, Shuhua

    2014-02-15

    A high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was optimised for simultaneous determination of fructose, sorbitol, glucose and sucrose in fruits. The analysis was carried out on a Phenomenex Luna 5u NH₂ 100A column (250 mm × 4.60mm, 5 micron) with isocratic elution of acetonitrile:water (82.5:17.5, v/v). Drift tube temperature of the ELSD system was set to 82 °C and nitrogen flow rate was 2.0 L min⁻¹. The regression equation revealed good linear relationship (R = 0.9967-0.9989) within test ranges. The limits of detection (LOD) and quantification (LOQ) for four analytes (peach, apple, watermelon, and cherry fruits) were in the range of 0.07-0.27 and 0.22-0.91 mg L⁻¹, respectively. The proposed HPLC-ELSD method was validated for quantification of sugars in peach, apple, watermelon, and cherry fruits, and the results were satisfactory. The results showed that the contents of the four sugars varied among fruits. While fructose (5.79-104.01 mg g⁻¹) and glucose (9.25-99.62 mg g⁻¹) emerged as common sugars in the four fruits, sorbitol (8.70-19.13 mg g⁻¹) were only found in peach, apple and cherry fruits, and sucrose (15.82-106.39 mg g⁻¹) were in peach, apple and watermelon. There was not detectable sorbitol in watermelon and sucrose in cherry fruits, respectively.

  14. 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.

  15. Proteasome activity and proteasome subunit transcripts in human spermatozoa separated by a discontinuous Percoll gradient.

    PubMed

    Rosales, O; Opazo, C; Diaz, E S; Villegas, J V; Sanchez, R; Morales, P

    2011-04-01

    Human semen is composed of a heterogeneous population of spermatozoa with varying degrees of structural and functional differentiation and normality, which result in subpopulations of different quality. Using a discontinuous Percoll gradient, we separated three subsets of spermatozoa (65/45%, 90/65% and 90% fractions) from normozoospermic semen samples from healthy donors and proceeded to characterise their morphology, viability, motility and proteasome activity. In addition, the presence of proteasome subunit transcripts was investigated using reverse transcription-polymerase chain reaction (RT-PCR). The results obtained showed significant differences in sperm motility, viability and morphology between the cells collected from each of the fractions. In particular, normal sperm morphology was 4.5 times higher in the 90% pellet in comparison with the 65/45% interface. In addition, there were significant differences in proteasomal activity between spermatozoa recovered from the 90% pellet and spermatozoa recovered from the 65/45% interface. Finally, there was a positive correlation between sperm proteasomal enzymatic activity and sperm motility and normal morphology after separation by a discontinuous Percoll gradient. The results of the RT-PCR revealed the presence of transcripts for the proteasome subunits β1, β2 and β5 in the human spermatozoa analysed. In conclusion, poor quality spermatozoa isolated from a Percoll gradient display an intrinsic proteasome activity deficiency, which may be associated with their low fertilising potential.

  16. 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

  17. Simultaneous determination of captopril and hydrochlorothiazide in human plasma by reverse-phase HPLC from linear gradient elution.

    PubMed

    Huang, Taomin; He, Zhong; Yang, Bei; Shao, Luping; Zheng, Xiaowei; Duan, Gengli

    2006-05-01

    A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of captopril and hydrochlorothiazide in human plasma samples was developed. Captopril was derivatized with 2,4'-dibromoacetophenone (pBPB) to form a captopril-pBPB adduct. From acidified serum plasma samples, the hydrochlorothiazide and derivatized captopril was extracted with 5 ml ether, then with 5 ml dichloromethane. Effective chromatographic separation was achieved using a C(18) column (DIAMONSIL 150 mmx4 mm i.d., 5 microm) based on an acetonitrile-trifluoroacetic acid-water gradient elution at a flow rate of 1.2 ml/min. The internal standard (IS), derivatized captopril and hydrochlorothiazide were detected at 263 nm and were eluted at 4.2, 6.8 and 16.9 min, respectively. No endogenous substances were found to interfere. The limit of quantification for hydrochlorothiazide and derivatized captopril in plasma were 3.3 and 7 ng/ml. The calibration curve for derivatized captopril showed linearity in the range 20-4000 ng/ml, with a regression coefficient corresponding to 0.9993 and the coefficient of the variation of the points of the calibration curve being lower than 10%. The calibration curve for hydrochlorothiazide showed linearity in the range 10-1200 ng/ml, with a regression coefficient corresponding to 0.9999 and the coefficient of the variation of the points of the calibration curve being lower than 10%. The method was suitably validated and successfully applied to the determination of captopril and hydrochlorothiazide in human plasma samples.

  18. A new affinity-HPLC packing for protein separation: Cibacron blue attached uniform porous poly(HEMA-co-EDM) beads.

    PubMed

    Unsal, Ender; Durdu, Aysun; Elmas, Begum; Tuncel, Murvet; Tuncel, Ali

    2005-11-01

    In this study, a new affinity high-performance liquid chromatography (HPLC) stationary phase suitable for protein separation was synthesized. In the first stage of the synthesis, uniform porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(HEMA-co-EDM), beads 6.2 mum in size were obtained. Homogeneous distribution of hydroxyl groups in the bead interior was confirmed by confocal laser scanning microscopy. The plain poly(HEMA-co-EDM) particles gave very low non-specific protein adsorption with albumin. The selected dye ligand Cibacron blue F3G-A (CB F3G-A) was covalently linked onto the beads via hydroxyl groups. In the batch experiments, albumin adsorption up to 60 mg BSA/g particles was obtained with the CB F3G-A carrying poly(HEMA-co-EDM) beads. The affinity-HPLC of selected proteins (albumin and lysozyme) was investigated in a 25 mm x 4.0-mm inner diameter column packed with CB F3G-A carrying beads and both proteins were successfully resolved. By a single injection, 200 mug of protein was loaded and quantitatively eluted from the column. The protein recovery increased with increasing flow rate and salt concentration of the elution buffer and decreased with the increasing protein feed concentration. During the albumin elution, theoretical plate numbers up to 30,000 plates/m were achieved by increasing the salt concentration.

  19. Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. III. Separation mechanism and gradient optimization.

    PubMed

    Mengerink, Y; Peters, R; van der Wal, Sj; Claessens, H A; Cramers, C A

    2002-03-01

    The first six linear and cyclic oligomers of polyamide-6 can be quantitatively determined in the polymer using HPLC with the sandwich injection method and an aqueous acetonitrile gradient. In this final part of the triptych concerning the determination of the oligomers in polyamide-6, the irregular elution behavior of the cyclic monomer compared to the cyclic oligomers was investigated. We also optimized the separation of the involved polyamide oligomers, with respect to gradient steepness, stationary phase, column temperature and mobile phase pH. The irregular elution behavior of the cyclic monomer could be caused by its relatively large exposed/accessible hydrophobic surface, which permits relatively easy penetration into the hydrophobic stationary phase giving extra retention. The dipole moment of the different oligomers was used as a measure for this exposed/accessible hydrophobic area to correlate the retention factors using quantitative structure-retention relationships. We also studied the retention behavior of the polyamide, which is injected each run directly onto the column and modifies the stationary phase. Using a 250-microl post gradient injection zone of formic acid on a 250x3 mm Zorbax SB-C18 column, the polyamide could be effectively removed from the stationary phase after each separation. The linear solvent strength (LSS) model was used to optimize the separation of the first six linear and cyclic oligomers. As the LSS model assumes a linear correlation between the modifier concentration and the logarithm of the retention factor and the cyclic monomer and dimer show extreme curvation of this relation in the eluting region, we investigated different models to predict gradient elution from isocratic data. A direct translation of the isocratic data to gradient retention times did not yield adequate retention times using the LSS model. It was found that the LSS model worked acceptably if gradient retention times were used as input data. Even for fast

  20. Mass spectrometry and NMR spectroscopy: modern high-end detectors for high resolution separation techniques--state of the art in natural product HPLC-MS, HPLC-NMR, and CE-MS hyphenations.

    PubMed

    Seger, Christoph; Sturm, Sonja; Stuppner, Hermann

    2013-07-01

    Current natural product research is unthinkable without the use of high resolution separation techniques as high performance liquid chromatography or capillary electrophoresis (HPLC or CE respectively) combined with mass spectrometers (MS) or nuclear magnetic resonance (NMR) spectrometers. These hyphenated instrumental analysis platforms (CE-MS, HPLC-MS or HPLC-NMR) are valuable tools for natural product de novo identification, as well as the authentication, distribution, and quantification of constituents in biogenic raw materials, natural medicines and biological materials obtained from model organisms, animals and humans. Moreover, metabolic profiling and metabolic fingerprinting applications can be addressed as well as pharmacodynamic and pharmacokinetic issues. This review provides an overview of latest technological developments, discusses the assets and drawbacks of the available hyphenation techniques, and describes typical analytical workflows.

  1. Automated HPLC separation of endohedral metallofullerene ScC{sub 2n} and YC{sub 2n} fractions

    SciTech Connect

    Stevenson, S.; Dorn, H.C.; Burbank, P.; Harich, K.; Haynes, J. Jr.; Kiang, C.H.; Salem, J.R.; DeVries, M.S.; Loosdrecht, P.H.M. van; Johnson, R.D.; Yannoni, C.S.; Bethune, D.S.

    1994-09-01

    We describe an automated HPLC separation of the endohedral metallofullerenes such as SCC{sub 2n} and YC{sub 2n} from empty-cage fullerenes utilizing two polystyrene chromatographic columns (500 and 1000 A) in series. Rapid separation of the metallofullerene fraction from the empty-cage fullerenes (e.g., C{sub 60}) under anaerobic conditions is achieved. For the isolated SCC{sub 2n} fraction, all even-carbon-membered species from Sc{sub 2}C{sub 74} to Sc{sub 2}C{sub 104} were identified by negative-ion chemical ionization mass spectrometry. In addition, Sc{sub 3}C{sub 82} was a prominent component of this fraction. For the separated YC{sub 2n} sample, the mass spectral data indicate the presence of YC{sub 82} and all even-carbon-numbered diyttrium species from Y{sub 2}C{sub 82} to Y{sub 2}C{sub 104}. 14 refs., 7 figs.

  2. Kinetic performance limits of constant pressure versus constant flow rate gradient elution separations. Part I: theory.

    PubMed

    Broeckhoven, K; Verstraeten, M; Choikhet, K; Dittmann, M; Witt, K; Desmet, G

    2011-02-25

    We report on a general theoretical assessment of the potential kinetic advantages of running LC gradient elution separations in the constant-pressure mode instead of in the customarily used constant-flow rate mode. Analytical calculations as well as numerical simulation results are presented. It is shown that, provided both modes are run with the same volume-based gradient program, the constant-pressure mode can potentially offer an identical separation selectivity (except from some small differences induced by the difference in pressure and viscous heating trajectory), but in a significantly shorter time. For a gradient running between 5 and 95% of organic modifier, the decrease in analysis time can be expected to be of the order of some 20% for both water-methanol and water-acetonitrile gradients, and only weakly depending on the value of V(G)/V₀ (or equivalently t(G)/t₀). Obviously, the gain will be smaller when the start and end composition lie closer to the viscosity maximum of the considered water-organic modifier system. The assumptions underlying the obtained results (no effects of pressure and temperature on the viscosity or retention coefficient) are critically reviewed, and can be inferred to only have a small effect on the general conclusions. It is also shown that, under the adopted assumptions, the kinetic plot theory also holds for operations where the flow rate varies with the time, as is the case for constant-pressure operation. Comparing both operation modes in a kinetic plot representing the maximal peak capacity versus time, it is theoretically predicted here that both modes can be expected to perform equally well in the fully C-term dominated regime (where H varies linearly with the flow rate), while the constant pressure mode is advantageous for all lower flow rates. Near the optimal flow rate, and for linear gradients running from 5 to 95% organic modifier, time gains of the order of some 20% can be expected (or 25-30% when accounting for

  3. 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.

  4. 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

  5. Rapid separation and analysis of six organic acids in Bayer liquors by RP-HPLC after solid-phase extraction.

    PubMed

    Xiao, Jian Bo; Chen, Xiao Qing; Jiang, Xin Yu; Wu, Sheng De

    2006-01-01

    A rapid revised phase high-performance liquid chromatographic (RP-HPLC) method for the determination of six organic acids in Bayer liquors is reported. Oxalic, tartaric, acetic, succinic, glutaric and butene dicarboxylic acid were separated and quantified in 10 min. First time repeatability, reproducibility and recoveries were determined out for these acids in Bayer liquors. The organic acids were removed from Bayer liquor by using a solid-phase extraction procedure with anion-exchange cartridges. The chromatographic separation was achieved with only one Kromasil RP-C18 column thermo stated at 25 degrees C. Organic acids were detected with a UV-vis detector (215 nm). The precision results showed that the relative standard deviations of the repeatability and reproducibility were < 2.80% and < 3.74%, respectively. The accuracy of the method was confirmed with an average recovery ranging between 85.2 and 107.3%. Under optimum conditions the detection limits ranged from 50 to 1000 mg/L.

  6. 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

  7. Setting up High Gradient Magnetic Separation for combating eutrophication of inland waters.

    PubMed

    Merino-Martos, A; de Vicente, J; Cruz-Pizarro, L; de Vicente, I

    2011-02-28

    To find new approaches to devise technologies for handling with eutrophication of inland waters is a global challenge. Separation of the P from water under conditions of continuous flow is proposed as an alternative and effective method. This work is based on using highly magnetic particles as the seeding adsorbent material and their later removal from solution by High Gradient Magnetic Separation (HGMS). Contrast to other methods based on batch conditions, large volumes of water can be easily handled by HGMS because of decreasing retention times. This study identifies the best working conditions for removing P from solution by investigating the effects of a set of four different experimental variables: sonication time, flow rate (as it determines the retention time of particles in the magnetic field), magnetic field strength and the iron (Fe) particles/P concentration ratio. Additionally, the change of P removal efficiency with time (build up effect) and the possibility of reusing magnetic particles were also studied. Our results evidenced that while flow rate does not significantly affect P removal efficiency in the range 0.08-0.36 mL s(-1), sonication time, magnetic field strength and the Fe particles/P concentration ratio are the main factors controlling magnetic separation process.

  8. Separation and characterization of oxaliplatin dinucleotides from DNA using HPLC-ESI ion trap mass spectrometry.

    PubMed

    Mowaka, Shereen; Linscheid, Michael

    2008-11-01

    Oxaliplatin is a third-generation platinum complex, and has a broad spectrum of antitumor activity. Such platinum complexes with the DACH carrier ligand have recently received increasing attention since they show efficacy against cisplatin-resistant cell lines. As the foremost indication of antitumor activity of platinum drugs is the formation of adducts with genomic DNA, calf thymus DNA-oxaliplatin adducts were the major target in this study. Calf thymus DNA was incubated with oxaliplatin, resulting in the formation of a large number of platinum-DNA adducts. Treated DNA was digested into the dinucleotides with a combination of enzymes, namely, benzonase, alkaline phosphatase, and nuclease S1. Using a high-performance liquid chromatography, we carried out the separation of individual platinum-DNA adducts which were concurrently identified using electrospray ionization ion trap mass spectrometry (MS). Both 1,2-intrastrand and 1,2-interstrand cross-linked adducts were found; however, those of the intrastrand nature have a considerably higher abundance than those of the interstrand cross-links. Among them, d(GpG)-oxaliplatin was the most abundant bifuctional adduct. To a lesser extent, a few monofunctional adducts were detected as well. MS(n) experiments served to ascertain the detailed structures of oxaliplatin adducts of dinucleoside monophosphates and of dinucleotides.

  9. R&D Isotope Separation with Large Thermal Gradients for Ultra-Low Background Experiments

    NASA Astrophysics Data System (ADS)

    Alanson Chiller, Angela; Chiller, Christopher; Jasinski, Benjamin; Snyder, Nathan; Mei, Dongming

    2013-04-01

    Seeking an alternative method to obtain enriched 76Ge and 73Ge for planned neutrinoless double-beta decay and dark matter experiments, a cryogenic distillation column is developed in our university laboratory for germane gas. Prior to experimenting with germane gas, which requires significant safety measures in place, the distillation column was tested with CO2. The preliminary analysis shows that 45CO2 was separated and enriched within two hours at room temperature. Exploiting the isotopic gradient within the two-meter length and removing preferentially from top and bottom points, 44CO2, 45CO2, and 46CO2 were bottled for further analysis. Methods of preferential condensation and vaporization used for depletion and enrichment of bottled gases show equal viability for purification and isotope enrichment with CO2 as well as gases with phase changes within the equipment specifications. Potential for purification and isotope enrichment/depletion of gases such as argon and xenon are investigated.

  10. 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

  11. Simultaneous multiresponse optimization of an HPLC method to separate seven cephalosporins in plasma and amniotic fluid: application to validation and quantification of cefepime, cefixime and cefoperazone.

    PubMed

    Nemutlu, Emirhan; Kir, Sedef; Katlan, Doruk; Beksaç, M Sinan

    2009-11-15

    An HPLC method for the separation of seven cephalosporins [Cefepime (CEP), ceftazidime (CTA), ceftizaxime (CTI), ceftriaxone (CTR), cefotaxime (COT), cefixime (CIX) and cefoperazone (COP)] in human plasma and amniotic fluid has been developed. Optimization of the chromatographic method was performed in three steps: a series of initial experiments followed by two sets of experiments based on different experimental designs. The initial experiments were performed to decide the basic analytical requirements of the method. Then screening experiment fractional factorial design was used in order to decrease the number of parameters by eliminating parameters which having insignificant effect on responses. The parameters having significant effect were further optimized through a full factorial design. Having studied two responses (retention times and resolutions), a desirability function that assess the responses together, was used to find experimental conditions where the system generated desirable results. The desirable results were obtained with XTerra C18 (250 mm x 4.6mm, 5 microm i.d.) column, 40 mM phosphate buffer, pH 3.2, 18% MeOH, 0.85 mL min(-1) flow rate and 32 degrees C column temperature. Gradient elution with MeOH was applied. A simple and efficient solid-phase extraction was applied for the preparation of plasma and amniotic fluid samples. The validation parameters of the method were evaluated in accordance with ICH guideline. The method validated was applied to the analysis of CEP and COP in maternal venous, fetal venous and fetal arterial plasma, and to the analysis of CIX in maternal venous plasma and amniotic fluid.

  12. 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

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

    PubMed

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

    2006-07-01

    Because of 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 nonporous-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 fmol 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.

  14. 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.

  15. Recovery of metallic copper by integrated chemical reduction and high gradient magnetic separation.

    PubMed

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

    2011-01-01

    The recovery of metals from waste effluents is necessary for pollution prevention and sustainable practice. High gradient magnetic separation (HGMS) is seen as a viable method. We investigated the capture of valence copper from aqueous copper ion by HGMS in combination with a chemical reduction process. When a copper solution (3.9 or 15.6 mM) was exposed to excess of dithionite (mole ratio of 1:3) in the presence of ammonia (mole ratio of 4) and amended with MnCl2 (2.5 g/L) and the mixture passed through a flow reactor under a strong magnetic field (10000 Gauss), valence copper was obtained and captured in the reactor with well over 95% yields. The chemical reduction reactions were unaffected by the presence of MnCl2 while the amount of MnCl2 (0, 20 and 32 mM) has significantly varied the copper recovery efficiency, especially in the case of high initial copper ion concentration (15.6 mM). Formation of MnO2 flocs was found to have a detrimental effect on copper removal efficiency. The HGMS method offers a tool of resource recovery for copper from waste effluents.

  16. Chemical interferences when using high gradient magnetic separation for phosphate removal: consequences for lake restoration.

    PubMed

    de Vicente, I; Merino-Martos, A; Guerrero, F; Amores, V; de Vicente, J

    2011-09-15

    A promising method for lake restoration is the treatment of lake inlets through the specific adsorption of phosphate (P) on strongly magnetizable particles (Fe) and their subsequent removal using in-flow high gradient magnetic separation (HGMS) techniques. In this work, we report an extensive investigation on the chemical interferences affecting P removal efficiencies in natural waters from 20 Mediterranean ponds and reservoirs. A set of three treatments were considered based on different Fe particles/P concentration ratios. High P removal efficiencies (>80%) were found in freshwater lakes (conductivities<600 μ S cm(-1)). However, a significant reduction in P removal was observed for extremely high mineralized waters. Correlation analysis showed that major cations (Mg(2+), Na(+) and K(+)) and anions (SO(4)(2-) and Cl(-)) played an essential role in P removal efficiency. Comparison between different treatments have shown that when increasing P and Fe concentrations at the same rate or when increasing Fe concentrations for a fixed P concentration, there exist systematic reductions in the slope of the regression lines relating P removal efficiency and the concentration of different chemical variables. These results evidence a general reduction in the chemical competition between P and other ions for adsorption sites on Fe particles. Additional analyses also revealed a reduction in water color, dissolved organic carbon (DOC) and reactive silicate (Si) concentrations with the addition of Fe microparticles.

  17. 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.

  18. On-line nano-HPLC/ESI QTOF MS and tandem MS for separation, detection, and structural elucidation of human erythrocytes neutral glycosphingolipid mixture.

    PubMed

    Kirsch, Stephan; Zarei, Mostafa; Cindrić, Mario; Müthing, Johannes; Bindila, Laura; Peter-Katalinić, Jasna

    2008-06-15

    A superior approach involving nano-high-performance liquid chromatography (nano-HPLC) in on-line conjunction to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI QTOF MS) and tandem MS for screening and structural characterization of complex mixtures of neutral glycosphingolipids (GSLs) is here described. Neutral GSLs purified from human erythrocytes were efficiently separated according to the differences in carbohydrate chain length by an optimized nano-HPLC protocol and flow-through detected by ESI QTOF MS at the low femtomole level. Additionally, GSL species were accurately distinguished from the accompanying lipids in the mixture, thus permitting the determination of detailed structural characteristics by data-dependent analysis for identification of GSL constitution within single experiments. An alternative nano-HPLC/ESI QTOF MS approach was designed for dissection of unsaturation/saturation degree of the ceramide moieties defining the hydrophobic portion of GSLs and subsequent localization by nano-HPLC/ESI QTOF MS/MS of the -CH=CH- within the ceramide regions. The method is fast, highly sensitive, and high-throughput amenable and is highlighted as a new and valuable analytical dimension in glycolipidomics.

  19. 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

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

    SciTech Connect

    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.

  1. 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.

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

    PubMed

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

    2013-11-29

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

  3. Superconducting open-gradient magnetic separation for the pretreatment of radioactive or mixed waste vitrification feeds. 1997 annual progress report

    SciTech Connect

    Doctor, R.; Nunez, L.; Cicero-Herman, C.A.; Ritter, J.A.; Landsberger, S.

    1997-01-01

    'Vitrification has been selected as a final waste form technology in the US for long-term storage of high-level radioactive wastes (HLW). However, a foreseeable problem during vitrification in some waste feed streams lies in the presence of elements (e.g., transition metals) in the HLW that may cause instabilities in the final glass product. The formation of spinel compounds, such as Fe{sub 3}O{sub 4} and FeCrO{sub 4}, results in glass phase separation and reduces vitrifier lifetime, and durability of the final waste form. A superconducting open gradient magnetic separation (OGMS) system maybe suitable for the removal of the deleterious transition elements (e.g. Fe, Co, and Ni) and other elements (lanthanides) from vitrification feed streams due to their ferromagnetic or paramagnetic nature. The OGMS systems are designed to deflect and collect paramagnetic minerals as they interact with a magnetic field gradient. This system has the potential to reduce the volume of HLW for vitrification and ensure a stable product. In order to design efficient OGMS and High gradient magnetic separation (HGMS) processes, a fundamental understanding of the physical and chemical properties of the waste feed streams is required. Using HLW simulant and radioactive fly ash and sludge samples from the Savannah River Technology Center, Rocky Flats site, and the Hanford reservation, several techniques were used to characterize and predict the separation capability for a superconducting OGMS system.'

  4. 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

  5. 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.

  6. 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.

  7. Binary electroosmotic-pump nanoflow gradient generator for miniaturized high-performance liquid chromatography.

    PubMed

    Zhou, Lei; Lu, Joann Juan; Gu, Congying; Liu, Shaorong

    2014-12-16

    High-performance liquid chromatography (HPLC) plays an important role in biotechnology, and a majority of chromatographic separations use gradient elution. While gradient generators can be built in different formats, binary pumps or quaternary pumps are most frequently used for gradient generator constructions. We have recently developed a high-pressure electroosmotic pump (EOP); the pump can be manufactured at a cost of a few hundred dollars. However, it is challenging to use this pump to deliver a gradient eluent directly. In this study, we first improve the monolith preparation by applying a pressure to the monomer solution during polymerization. We assemble a binary EOP gradient generator and discuss the relationship between the gradient profile and voltage applied to the EOP. We demonstrate the feasibility of the binary EOP gradient generator for generating a smooth and reproducible nanoflow gradient. After integration of the gradient generator into a miniaturized HPLC system, we use the HPLC system for separating peptide mixtures from trypsin-digested proteins. The performance comparison between the above miniaturized HPLC system and an Agilent 1200 HPLC system exhibits comparable efficiencies, resolutions, and peak capacities.

  8. Phase separation of binary mixtures in thin films: Effects of an initial concentration gradient across the film.

    PubMed

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

    2012-04-01

    We study the kinetics of phase separation of a binary (A,B) mixture confined in a thin film of thickness D by numerical simulations of the corresponding Cahn-Hilliard-Cook (CHC) model. The initial state consisted of 50% A:50% B with a concentration gradient across the film, i.e., the average order parameter profile is Ψav(z,t=0)=(2z/D-1)Ψg,0≤z≤D, for various choices of Ψg and D. The equilibrium state (for time t→∞) consists of coexisting A-rich and B-rich domains separated by interfaces oriented perpendicular to the surfaces. However, for sufficiently large Ψg, a (metastable) layered state is formed with a single interface parallel to the surfaces. This phenomenon is explained in terms of a competition between domain growth in the bulk and surface-directed spinodal decomposition (SDSD) that is caused by the gradient. Thus, gradients in the initial state can stabilize thin-film morphologies which are not stable in full equilibrium. This offers interesting possibilities as a method for preparing novel materials. PMID:22680483

  9. 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

  10. Research on high gradient magnetic separation of pneumatic conveyed powder products: Investigation from the viewpoint of interparticle interactions

    NASA Astrophysics Data System (ADS)

    Senkawa, Kohei; Nakai, Yuki; Mishima, Fumihito; Akiyama, Yoko; Nishijima, Shigehiro

    2013-01-01

    The separation and removal of the metallic debris originating from pipe of manufacturing line are required in the manufacturing process of the fine particle products. In this study, we develop a high gradient magnetic separation system (HGMS) under a dry process by using a superconducting magnet to remove ferromagnetic particles such as the material stainless steel (SUS). To avoid the obstruction of the separation part by aggregation of the processed material, we develop a magnetic separation system using a pneumatic conveying as a new transportation method of the particles. The magnetic separations were experimented under the same conditions on different days, but the results were different. The reason is considered to be the difference in adhesion force between the particles due to a change of humidity, we have measured the adhesion forces between the ferromagnetic particles and the paramagnetic medium particles using AFM (Atomic Force Microscope) while changing the humidity. As a result, the adhesion force between the particles increased with the increasing of humidity. Furthermore, we saw that the effect of relative humidity was larger in the adhesion force of alumina with larger cohesive property. Based on these results, an appropriate condition of the separation experiment was clarified. And a dehumidification mechanism was introduced.

  11. 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.

  12. Concentration gradient focusing and separation in a silica nanofluidic channel with a non-uniform electroosmotic flow.

    PubMed

    Hsu, Wei-Lun; Harvie, Dalton J E; Davidson, Malcolm R; Jeong, Helen; Goldys, Ewa M; Inglis, David W

    2014-09-21

    The simultaneous concentration gradient focusing and separation of proteins in a silica nanofluidic channel of various geometries is investigated experimentally and theoretically. Previous modelling of a similar device [Inglis et al., Angew. Chem. Int. Ed., 2011, 50, 7546] assumed a uniform velocity profile along the length of the nanochannel. Using detailed numerical analysis incorporating charge regulation and viscoelectric effects, we show that in reality the varying axial electric field and varying electric double layer thickness caused by the concentration gradient, induce a highly non-uniform velocity profile, fundamentally altering the protein trapping mechanism: the direction of the local electroosmotic flow reverses and two local vortices are formed near the centreline of the nanochannel at the low salt concentration end, enhancing trapping efficiency. Simulation results for yellow/red fluorescent protein R-PE concentration enhancement, peak focusing position and peak focusing width are in good agreement with experimental measurements, validating the model. The predicted separation of yellow/red (R-PE) from green (Dyl-Strep) fluorescent proteins mimics that from a previous experiment [Inglis et al., Angew. Chem. Int. Ed., 2011, 50, 7546] conducted in a slightly different geometry. The results will inform the design of new class of matrix-free particle focusing and separation devices. PMID:25027204

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

    PubMed

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

    2010-03-01

    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.

  14. 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

  15. 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

  16. Optical activity and ultraviolet absorbance detection of dansyl L-amino acids separated by gradient liquid chromatography

    SciTech Connect

    Not Available

    1987-04-01

    Many scientific investigations (e.g., geochronology, pharmaceuticals) have the need to determine enantiometric ratios of amino acids and other compounds. It has been reported that OA/UV or OA/RI (refractive index) are ideal methods for the determination of enantiomeric ratios without the need for chiral columns, chiral eluents, or diasteromer preparation. Unfortunately, only three amino acids are naturally UV absorbing (254 nm), and RI sensitivity for amino acids is low. Derivatization by several methods (o-phthalaldehyde, dansyl, phenylisothiocyanate, fluorescamine, 2,4-dinitrofluorobenzene, and phenylthiohydantoin) renders all amino acids UV absorbing and makes UV or fluorescence viable techniques for amino acids determinations. A previously neglected aspect of derivatization is the effect on optical activity. These highly polar groups influence the chiral center of amino acids drastically (electronic and steric effects). The shifting of the absorption band to the proximity of the wavelength used for OA measurements further enhances the importance of the substituent. The authors report here the determination of 17 dansyl amino acids in a mixture by UV absorbance and optical activity. This involves gradient elution. Previously, the optical activity detector (OAD) has been used only with isocratic HPLC.

  17. 2D modeling and preliminary in vitro investigation of a prototype high gradient magnetic separator for biomedical applications.

    PubMed

    Chen, Haitao; Kaminski, Michael D; Rosengart, Axel J

    2008-01-01

    High gradient magnetic separation (HGMS) of magnetic materials from fluids or waste products has many established industrial applications. However, there is currently no technology employing HGMS for ex-vivo biomedical applications, such as for the removal of magnetic drug- or toxin-loaded spheres from the human blood stream. Importantly, human HGMS applications require special design modifications as, in contrast to conventional use where magnetic elements are permanently imbedded within the separation chambers, medical separators need to avoid direct contact between the magnetic materials and blood to reduce the risk of blood clotting and to facilitate convenient and safe treatment access for many individuals. We describe and investigate the performance of a magnetic separator prototype designed for biomedical applications. First, the capture efficiency of a prototype HGMS separator unit consisting of a short tubing segment and two opposing magnetizable fine wires along the outside of the tubing was investigated using 2D mathematical modeling. Second, the first-pass effectiveness to remove commercially available, magnetic polystyrene spheres from human blood using a single separator unit was experimentally verified. The theoretical and experimental data correlated well at low flow velocities (<5.0 cm/s) and high external magnetic fields (>0.05 T). This prototype separator unit removed >90% in a single pass of the magnetic spheres from water at mean flow velocity < or =8.0 cm/s and from blood mimic fluids (ethylene glycol-water solutions) at mean flow velocity < or =2.0 cm/s. In summary, we describe and prove the feasibility of a HGMS separator for biomedical applications.

  18. Human-mouse mixed lymphocyte cultures. II. Partial separation of functionally distinct populations on discontinuous albumin gradients.

    PubMed Central

    Boylston, A W; Anderson, R L

    1979-01-01

    Human-mouse mixed lymphocyte cultures (MLC) develop stable, strain-specific responses directed towards antigens determined by the mouse major histocompatibility complex (MHC). By restimulation in vitro a two- to four-fold increase in total cell numbers can be achieved. Sensitized cells can be fractionated on discontinuous BSA gradients to produce fractions with predominantly proliferative or cytotoxic activity towards the intiating antigens. Mixing experiments show that fractionation of biological activity is the result of fractination of specifically sensitized effector cells rather than fractionation of inhibitory or collaborative elements. Since biological activities or can be separated on the basis of physical properties into distinct cell populations these experiments support the idea that these functions are the properties of distinct subclasses of human T lymphocyte. Xenogeneic MLC coupled to physical separation measures is a useful approach to the study of antigen-specific human T lymphocytes. PMID:155651

  19. Human-mouse mixed lymphocyte cultures. II. Partial separation of functionally distinct populations on discontinuous albumin gradients.

    PubMed

    Boylston, A W; Anderson, R L

    1979-02-01

    Human-mouse mixed lymphocyte cultures (MLC) develop stable, strain-specific responses directed towards antigens determined by the mouse major histocompatibility complex (MHC). By restimulation in vitro a two- to four-fold increase in total cell numbers can be achieved. Sensitized cells can be fractionated on discontinuous BSA gradients to produce fractions with predominantly proliferative or cytotoxic activity towards the intiating antigens. Mixing experiments show that fractionation of biological activity is the result of fractination of specifically sensitized effector cells rather than fractionation of inhibitory or collaborative elements. Since biological activities or can be separated on the basis of physical properties into distinct cell populations these experiments support the idea that these functions are the properties of distinct subclasses of human T lymphocyte. Xenogeneic MLC coupled to physical separation measures is a useful approach to the study of antigen-specific human T lymphocytes.

  20. 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

  1. Rapid separation of desloratadine and related compounds in solid pharmaceutical formulation using gradient ion-pair chromatography.

    PubMed

    Zheng, Jinjian; Rustum, Abu M

    2010-01-01

    We reported the development of an ion-pair chromatographic method to separate desloratadine and all known related compounds in Clarinex Tablets, which use desloratadine as active pharmaceutical ingredient (API). For the first time, baseline separation for desloratadine and all known related compounds was achieved by utilizing a YMC-Pack Pro C(18) column (150 mm x 4.6 mm I.D., 3 microm particle size, 120A pore size) and a gradient elution method. The mobile phase A contains 3 mM sodium dodecylsulfate (SDS), 15 mM sodium citrate buffer at pH 6.2, and 40 mM sodium sulfate, while the mobile phase B is acetonitrile. Chromsword, an artificial intelligence method development tool, was used to optimize several key chromatographic parameters simultaneously including buffer pH/solvent strength, and temperature/gradient profile. The resolution of desloratadine and desloratadine 3,4-dehydropiperidine derivative, one of the critical pairs was improved by adding 40 mM sodium sulfate. Ultraviolet detection at 267 nm was used to achieve the detection for desloratadine and all compounds. This method has been successfully validated according to ICH guidelines in terms of linearity, accuracy, quantitation limit/detection limit, precision, specificity and robustness. It could be used as a stability indicating method for desloratadine drug substances or drug products that use desloratadine as active pharmaceutical ingredient.

  2. 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.

  3. Simplified gradient generator for micro- and nano-liquid chromatography.

    PubMed

    Murata, Kaoru; Mano, Nariyasu; Asakawa, Naoki; Ishihama, Yasushi

    2006-08-01

    We have developed a simple device to generate gradient elution profiles using split tubing array (STAR) units for micro- and nano-HPLC. This gradient device consists of a delivery pump, a splitter, tubes in parallel, and a relatively large-volume mixing chamber. In the mixing chamber, an initially filled water-rich solvent is adjusted to an organic-rich solvent by delivery of appropriate components through the split paths, in which the flow rate and residence time are controlled by the sizes of the tubes employed. A program was developed to describe the output gradient profiles, and the predicted gradient profiles were highly consistent with the observed ones. A linear gradient generated by this STAR system was successfully applied to micro-HPLC systems for separation of digested peptides derived from serum albumin with sufficient reproducibility. Further miniaturization of STAR systems for nano-HPLC coupled with tandem mass spectrometry was accomplished to separate digested peptides from serum albumin with a reproducibility of retention times of better than 0.2%, and the obtained spectra from the well-separated chromatographic peaks allowed protein identification with high confidence by protein database searching. We believe that this simple and reproducible gradient system using an isocratic pump will be especially advantageous for nanoLC-MS, instead of flow-controlled gradient pumps.

  4. Separation and purification of bioactive botrallin and TMC-264 by a combination of HSCCC and semi-preparative HPLC from endophytic fungus Hyalodendriella sp. Ponipodef12.

    PubMed

    Mao, Ziling; Luo, Ruiya; Luo, Haiyu; Tian, Jin; Liu, Hongwei; Yue, Yang; Wang, Mingan; Peng, Youliang; Zhou, Ligang

    2014-09-01

    Two dibenzo-α-pyrones, botrallin (1) and TMC-264 (2) were preparatively separated from crude ethyl acetate extract of the endophytic fungus Hyalodendriella sp. Ponipodef12, which was isolated from the hybrid 'Neva' of Populus deltoides Marsh × P. nigra L. using a combination of high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC. Botrallin (1) with 74.73% of purity and TMC-264 (2) with 82.29% of purity were obtained through HSCCC by employing a solvent system containing n-hexane-ethyl acetate-methanol-water at a volume ratio of 1.2:1.0:0.9:1.0. It was the first time for TMC-264 (2) to be isolated from this fungus. TMC-264 (2) showed strong antimicrobial and antinematodal activity, and botrallin (1) exhibited moderate inhibitory activity on acetylcholinesterase.

  5. A new chiral derivatizing agent for the HPLC separation of α-amino acids on a standard reverse-phase column.

    PubMed

    Kotthaus, A F; Altenbach, H-J

    2011-02-01

    A new chiral derivatizing agent for α-amino acids is described which leads to diastereomers that can be separated by reverse-phase HPLC with direct detection by a diode array detector. The main advantage of the presented procedure is the fact that an excess of the derivatizing reagent can be employed as the product exhibits an absorption maximum at 360 nm, while the reagent has its absorption maximum at 260 nm. Therefore, it is possible to suppress the reagent signal by a detection wavelength of 400 nm leading to an easy and general method for the enantioseparation of a mixture of DL-amino acids and the determination of the enantiomeric purity of α-amino acid as exemplified by 16 different α-amino acids.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. [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

  12. 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

  13. Recent advances in SPE-chiral-HPLC methods for enantiomeric separation of chiral drugs in biological samples.

    PubMed

    Ali, Imran; Alam, Syed Dilshad; Al-Othman, Zeid A; Farooqi, Javed A

    2013-08-01

    In medical practices, the determination of enantiomeric ratio of the chiral drugs is very important for their activities, bioavailabilities and biodegradation. Only homochiral medication is safe for humans. The chiral analysis in biological samples is the first and most important step. The present article describes the technical strategies of the enantiomeric resolution of racemic drugs in biological samples. Attempts have been made to describe sample preparation by solid-phase extraction and enantiomeric resolution by chiral high-performance liquid chromatography. Various chiral stationary phases used in chiral separations of racemic drugs have been described. Efforts are also made to discuss the chiral recognition mechanism and future perspectives of chiral analyses in biological samples.

  14. Rapid separation and determination of process-related substances of paracetamol using reversed-phase HPLC with photo diode array as a detector.

    PubMed

    Rao, R Nageswara; Narasaraju, A

    2006-02-01

    A simple and rapid gradient reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of paracetamol and its related compounds in bulk drugs and pharmaceutical formulations has been developed. As many as nine process impurities and one degradation product of paracetamol have been separated on a Symmetry C18 column (4.6 x 250 mm i.d., particle size 5 microm) with gradient elution using 0.01 M potassium dihydrogen phosphate buffer (pH 3.0) and acetonitrile as mobile phase and photo diode array detection at 215 nm. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH values. The correlation coefficients for calibration curves for paracetamol as well as impurities were in the range of 0.9951 - 0.9994. The proposed RP-LC method was successfully applied to the analysis of commercial formulations; the recoveries of paracetamol were in the range of 99-101%. The method could be of use not only for rapid and routine evaluation of the quality of paracetamol in bulk drug manufacturing units but also for detection of its impurities in pharmaceutical formulations.

  15. 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.

  16. 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

  17. 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.

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

    PubMed

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

    2014-01-17

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

  19. 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

  20. Molecularly imprinted SPE coupled with HPLC for the selective separation and enrichment of alkyl imidazolium ionic liquids in environmental water samples.

    PubMed

    Xia, Gao; Jing, Fan; Guifen, Zhu; Xiaolong, Wang; Jianji, Wang

    2013-10-01

    A novel 1-butyl-3-methylimidazolium chloride ionic liquid surface imprinted solid-phase sorbent was synthesized. The as-prepared material was characterized by SEM, Brunauer-Emmett-Teller surface area analysis and Fourier Transform IR measurements. Then its adsorption properties for alkyl imidazolium ionic liquids, including adsorption capacities, adsorption kinetics, and properties of selective separation and enrichment were studied in detail. It was shown that the ionic liquid surface imprinted polymer exhibited high selective recognition characteristics for the imidazolium chloride ionic liquids with short alkyl chains (C(n)mimCl, n = 2, 4, 6, 8) and the adsorption equilibrium was achieved within 25 min. Various parameters were optimized for the 1-butyl-3-methylimidazolium chloride ionic liquid surface imprinted polymer SPE column, such as flow rate, eluent solvent, selectivity, and reusability of the column. Then, the SPE column coupled with HPLC was used for the determination of alkyl imidazolium ionic liquids. Experimental results showed that the existence of their structural analogs and common concomitants in environmental matrices did not affect the enrichment of 1-butyl-3-methyl imidazolium chloride ionic liquid. The average recoveries of 1-butyl-3-methylimidazolium chloride ionic liquid in spiked water samples were in the range of 92.0-102.0% with the RSD lower than 5.8%.

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

    PubMed

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

    2014-01-01

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

  2. 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

  3. 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.

  4. Separation of human lymphocytes on Ficoll-Paque gradients: stimulation of cells and depletion of a concanavalin-A responsive radioresistant subpopulation

    SciTech Connect

    Kol, R.; Friedlaender, M.; Riklis, E.; Raveh, D.

    1983-07-01

    A subpopulation of human lymphocytes separated on Ficoll-Paque gradients showed an ultrastructural phenotype characteristic of stimulated cells. Thymidine incorporation was increased fivefold compared with unseparated (Buffy coat) controls. The Ficoll-Paque separated lymphocytes were more sensitive to gamma radiation than unseparated lymphocytes and showed a decreased capability to undergo transformation in response to concanavalin A. Transformation in response to phytohemagglutinin and pokeweed mitogen was the same as for unseparated control lymphocytes. These results are interpreted as the selective depletion of a Con A-responsive T-cell fraction by Ficoll-Paque separation.

  5. Separation of human lymphocytes on Ficoll-Paque gradients: stimulation of cells and depletion of a concanavalin-A responsive radioresistant subpopulation

    SciTech Connect

    Kol, R.; Friedlaender, M.; Riklis, E.; Raveh, D.

    1983-07-01

    A subpopulation of human lymphocytes separated on Ficoll-Paque gradients showed an ultrastructural phenotype characteristic of stimulated cells. Thymidine incorporation was increased fivefold compared with unseparated (Buffy coat) controls. The Ficoll-Paque separated lymphocytes were more sensitive to ..gamma.. radiation than unseparated lymphocytes and showed a decreased capability to undergo transformation in response to concanavalin A. Transformation in response to phytohemagglutinin and pokeweed mitogen was the same as for unseparated control lymphocytes. These results are interpreted as the selective depletion of a Con A-responsive T-cell fraction by Ficoll-Paque separation.

  6. Characterization and quantitative determination of impurities in piperaquine phosphate by HPLC and LC/MS/MS.

    PubMed

    Dongre, Vaijanath G; Karmuse, Pravin P; Ghugare, Pradeep D; Gupta, Mukesh; Nerurkar, Bipin; Shaha, Chirag; Kumar, Ashok

    2007-01-01

    Four impurities in piperaquine phosphate bulk drug substance were detected by a newly developed gradient reverse phase high performance liquid chromatographic (HPLC) method. These impurities were identified by LC/MS/MS. The structures of impurities were confirmed by spectroscopic studies (NMR and IR) conducted using synthesized authentic compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. The system suitability of HPLC analysis established the validity of the separation. The method was validated according to ICH guidelines with respect to specificity, precision, accuracy and linearity. Forced degradation studies were also performed for piperaquine phosphate bulk drug samples to demonstrate the stability indicating power of the newly developed HPLC method. PMID:16916594

  7. 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.

  8. A rapid technique for lymphocyte preparation prior to two-color immunofluorescence analysis of lymphocyte subsets using flow cytometry. Comparison with density gradient separation.

    PubMed

    Mansour, I; Bourin, P; Rouger, P; Doinel, C

    1990-02-20

    A technique is described for lymphocyte preparation which permits analyses by two-color immunofluorescence and flow cytometry. It consists, briefly, of the lysis of red blood cells and washing of white blood cells prior to labeling. We tested this technique with a large panel of monoclonal antibodies in mono- and dual immunofluorescence. By comparing these results to those obtained after density gradient separation, we found the following statistically significant differences: the count of the phenotype B1+ was higher after whole blood lysis preparation than after density gradient separation; whereas, the corresponding counts of OKT4+ and Leu-4-Leu-7+ phenotypes were lower. No difference was detected with OKT8+, Leu-4+, OKT8+Leu-4+, OKT8+Leu-4-, OKT8-Leu-4+, OKT8+Leu-7+, Leu-4+Leu-7+, Leu-4-Leu-11c+, OKT8+Leu-11c+ and OKT8+Leu-15+ phenotypes. We have studied the reproducibility of both methods and the correlation between them. The disparity of the lymphocyte subset count between these two methods, though statistically significant, was relatively weak and seems to be due to the density gradient separation. Since the preparation of lymphocytes using the density gradient method is time consuming, we propose whole blood lysis as an alternative lymphocyte separation method when assessing immune status in human disease by flow cytometry. It offers the following advantages: (i) it does not require additional steps, (ii) it permits two-color immunofluorescence through the labeling of white blood cells after washing, (iii) it is reliable, (iv) it is reproducible, and (v) it is helpful in studies of lymphopenia since it offers the possibility of lymphocyte enrichment.

  9. One-step column chromatographic extraction with gradient elution followed by automatic separation of volatiles, flavonoids and polysaccharides from Citrus grandis.

    PubMed

    Han, Han-Bing; Li, Hui; Hao, Rui-Lin; Chen, Ya-Fei; Ni, He; Li, Hai-Hang

    2014-02-15

    Citrus grandis Tomentosa is widely used in traditional Chinese medicine and health foods. Its functional components include volatiles, flavonoids and polysaccharides which cannot be effectively extracted through traditional methods. A column chromatographic extraction with gradient elution was developed for one-step extraction of all bioactive substances from C. grandis. Dried material was loaded into a column with petroleum ether: ethanol (8:2, PE) and sequentially eluted with 2-fold PE, 3-fold ethanol: water (6:4) and 8-fold water. The elutes was separated into an ether fraction containing volatiles and an ethanol-water fraction containing flavonoids and polysaccharides. The later was separated into flavonoids and polysaccharides by 80% ethanol precipitation of polysaccharides. Through this procedure, volatiles, flavonoids and polysaccharides in C. grandis were simultaneously extracted at 98% extraction rates and simply separated at higher than 95% recovery rates. The method provides a simple and high-efficient extraction and separation of wide range bioactive substances.

  10. Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: salt gradient approach.

    PubMed

    Fekete, Szabolcs; Beck, Alain; Fekete, Jenő; Guillarme, Davy

    2015-01-01

    Ion exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge variants. When applying salt gradient IEX approach for monoclonal antibodies (mAbs) characterization, this approach is described as time-consuming to develop and product-specific. The goal of this study was to tackle these two bottle-necks. By modeling the retention of several commercial mAbs and their variants in IEX, we proved that the mobile phase temperature was not relevant for tuning selectivity, while optimal salt gradient program can be easily found based on only two initial gradients of different slopes. Last but not least, the dependence of retention vs. pH being polynomial, three initial runs at different pH were required to optimize mobile phase pH. Finally, only 9h of initial experiments were necessary to simultaneously optimize salt gradient profile and pH in IEX. The data can then be treated with commercial modeling software to find out the optimal conditions to be used, and accuracy of retention times prediction was excellent (less than 1% variation between predicted and experimental values). Second, we also proved that generic IEX conditions can be applied for the characterization of mAbs possessing a wide range of pI, from 6.7 to 9.1. For this purpose, a strong cation exchange column has to be employed at a pH below 6 and using a proportion of NaCl up to 0.2M. Under these conditions, all the mAbs were properly eluted from the column. Therefore, salt gradient CEX can be considered as a generic multi-product approach. PMID:25240157

  11. 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.

  12. Combination of preparative HPLC and HSCCC methods to separate phosphodiesterase inhibitors from Eucommia ulmoides bark guided by ultrafiltration-based ligand screening.

    PubMed

    Shi, Shu-Yun; Peng, Mi-Jun; Zhang, Yu-Ping; Peng, Sheng

    2013-05-01

    Phosphodiesterase (PDE) inhibitors are widely used because of their various pharmacological properties, and natural products are considered the most productive source of PDE inhibitors. In this work, a new ultrafiltration-high-performance liquid chromatography (HPLC)-diode-array detection-mass spectrometry based ligand screening was developed for the first screening of PDE inhibitors from Eucommia ulmoides bark, and then the target bioactive compounds were prepared by combination of stepwise preparative HPLC and high-speed countercurrent chromatography (HSCCC) methods. Experiments were conducted to optimize the parameters in ultrafiltration, stepwise preparative HPLC, and HSCCC to allow rapid and effective screening and isolation of active compounds from complex mixtures. Seven lignans with purity over 97 % were isolated and identified by their UV, electrospray ionization mass spectrometry, and NMR data as (+)-pinoresinol-4,4'-di-O-β-D-glucopyranoside (1), (+)-pinoresinol-4-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (2), (+)-medioresinol-4,4'-di-O-β-D-glucopyranoside (3), (+)-syringaresinol-4,4'-di-O- β-D-glucopyranoside (4), (-)-olivil-4'-O-β-D-glucopyranoside (5), (-)-olivil-4-O-β-D- glucopyranoside (6), and (+)-pinoresinol-4-O-β-D-glucopyranoside (7). Compound 2 was first isolated from the genus Eucommia. Lignan diglucopyranosides (compounds 1-4) shower a greater inhibitory effect than lignan monoglucopyranosides (compounds 5-7). The method developed could be widely applied for high-throughput screening and preparative isolation of PDE inhibitors from natural products.

  13. Development of HPLC/ESI-MS and HPLC/1H NMR methods for the identification of photocatalytic degradation products of iodosulfuron.

    PubMed

    Sleiman, Mohamad; Ferronato, Corinne; Fenet, Bernard; Baudot, Robert; Jaber, Farouk; Chovelon, Jean-Marc

    2006-05-01

    In the present study, HPLC/ESI-MS and stopped-flow HPLC/1H NMR methods were developed and applied to separate and characterize the byproducts arising from TiO2-catalyzed photodegradation of the herbicide iodosulfuron methyl ester (IOME) in aqueous solution under UV irradiation. Prior to identification, irradiated solutions of IOME (200 and 1000 mg.L(-1)) were concentrated by solid-phase extraction using two cartridges: Isolute C18 and Isolute ENV+. Analytical separation was achieved on a C18 reversed-phase column with ACN/H2O (HPLC/MS) or ACN/D2O (HPLC/NMR) as mobile phase and a linear gradient with a chromatographic run time of 35 min. The combination of UV and MS data allowed the structural elucidation of more than 20 degradation products, whereas 1H NMR data permitted an unequivocal confirmation of the identities of major products and the differentiation of several positional isomers, in particular, the hydroxylation isomers. The obtained results permitted us to propose a possible degradation scheme and to put in evidence the presence of privileged sites for the attack of OH radicals. This work shows, for the first time, the application of combined HPLC with UV, MS, and NMR detection for complete structural elucidation of photocatalytic degradation products, and it will be of particular value in studies on the elimination of pollutants in aqueous solutions by photocatalysis.

  14. 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.

  15. 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

  16. 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

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

    DOE PAGES

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

    2016-04-07

    We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Enantiomeric separations of illicit drugs and controlled substances using cyclofructan-based (LARIHC) and cyclobond I 2000 RSP HPLC chiral stationary phases.

    PubMed

    Padivitage, Nilusha L T; Dodbiba, Edra; Breitbach, Zachary S; Armstrong, Daniel W

    2014-06-01

    Recently a novel class of chiral stationary phases (CSPs) based on cyclofructan (CF) has been developed. Cyclofructans are cyclic oligosaccharides that possess a crown ether core and pendent fructofuranose moieties. Herein, we evaluate the applicability of these novel CSPs for the enantiomeric separation of chiral illicit drugs and controlled substances directly without any derivatization. A set of 20 racemic compounds were used to evaluate these columns including 8 primary amines, 5 secondary amines, and 7 tertiary amines. Of the new cyclofructan-based LARIHC columns, 14 enantiomeric separations were obtained including 7 baseline and 7 partial separations. The LARIHC CF6-P column proved to be the most useful in separating illicit drugs and controlled substances accounting for 11 of the 14 optimized separations. The polar organic mode containing small amounts of methanol in acetonitrile was the most useful solvent system for the LARIHC CF6-P CSP. Furthermore, the LARIHC CF7-DMP CSP proved to be valuable for the separation of the tested chiral drugs resulting in four of the optimized enantiomeric separations, whereas the CF6-RN did not yield any optimum separations. The broad selectivity of the LARIHC CF7-DMP CSP is evident as it separated primary, secondary and tertiary amine containing chiral drugs. The compounds that were partially or un-separated using the cyclofructan based columns were screened with a Cyclobond I 2000 RSP column. This CSP provided three baseline and six partial separations.

  3. 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.

  4. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients.

    PubMed Central

    Heuer, H; Krsek, M; Baker, P; Smalla, K; Wellington, E M

    1997-01-01

    A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils. PMID:9251210

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

    PubMed

    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.

  6. 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

  7. 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.

  8. Synthesis of. beta. -D-glucan in vitro: HPLC separation of oligosaccharides from enzymic digests of glucans synthesized by Golgi apparatus and UDP-glucose

    SciTech Connect

    Gibeaut, D.M.; Carpita, N.C. )

    1990-05-01

    Anion exchange HPLC (Dionex) resolves {beta}-D-glucose oligosaccharides which vary in (1{yields}3) and (1{yields}4) linkage structure. The products from incubation of UDPG and cellular membranes from maize enriched for Golgi apparatus, ER, and plasma membrane are digested with glucan hydrolases specific for either (1{yields}3) or (1{yields}4) linkages and one which requires a specific (1{yields}3){beta}-D-glc-(1{yields}4){beta}-D-glc sequence. Two oligomers released by the latter enzyme are diagnostic of cereal grass mixed-linkage {beta}-D-glucan and are used as standards to examine reaction conditions for synthesis of this polymer with radioactive substrates. Our preliminary data indicate that formation of specific sequences of {beta}-D-glucan is increased by high concentrations of UDPG. Other radioactive oligomers are present in greater quantity than those from authentic mixed-linkage glucan and their linkage structure is now under investigation.

  9. 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

  10. Application of stepwise gradients in counter-current chromatography: a rapid and economical strategy for the one-step separation of eight coumarins from Seseli resinosum.

    PubMed

    Shehzad, Omer; Khan, Salman; Ha, In Jin; Park, Youmie; Tosun, Alev; Kim, Yeong Shik

    2013-10-01

    The targeted purification of compounds with a broad polarity range from traditional medicinal plants is a big challenge for counter-current chromatography (CCC). Gradient elution was introduced in CCC to address this problem. However, once a suitable solvent system is selected, the separation process requires optimization of operational parameters. The present study was conducted to optimize various operational parameters to integrate the flow rate and solvent gradients for the rapid isolation of eight coumarins from Seseli resinosum in a single run. An increase in the system temperature from 15°C to 35°C increased the stationary phase retention and solubility of the sample, whereas the operation time and viscosity of the system were decreased. The high purity of each compound was ensured by collecting the fractions from the main peaks while all the shoulder peaks were mixed and separated under the same conditions with semi-preparative CCC. GC-FID was used to analyze the components of each phase, which was prepared without presaturation to save the time and solvent consumption. Finally, eight coumarins were purified, including (1) d-laserpitin, (2) (3'S,4'S)-3'-angeloyloxy-4'-hydroxy-3',4'-dihydroseselin, (3) (+)-samidin, (4) (3'S,4'S)-3'-acetoxy-4'-angeloyloxy-3',4'-dihydroseselin, (5) deltoin (6), calipteryxin, (7) (3'S,4'S)-3',4'-disenecioyloxy-3',4'-dihydroseselin, and (8) (-)-anomalin. The present technique has successfully accomplished the goal of one-step separation of these compounds with high purity and recovery in an economic and time efficient manner. PMID:23992840

  11. 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.

  12. 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

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2015-02-01

    Betalains, natural plant pigments, are beneficial compounds due to their antioxidant and possible chemoprotective properties. A mixture of betalains: betanin/isobetanin, decarboxybetanins and neobetanin from processed red beet roots (Beta vulgaris L.) juice was separated in food-grade, gradient solvent systems using high-performance counter-current chromatography (HPCCC). The decarboxylated and dehydrogenated betanins were obtained by thermal degradation of betanin/isobetanin from processed B. vulgaris L. juice under mild conditions. Two solvent systems (differing in their composition by phosphoric acid and ethanol volume gradient) consisting of BuOH-EtOH-NaClsolution-H2O-H3PO4 (v/v/v/v/v, 1300:200-1000:1300:700:2.5-10) in the 'tail-to-head' mode were run. The flow rate of the mobile phase (organic phase) was 1.0 or 2.0 ml/min and the column rotation speed was 1,600 rpm (20°C). The retention of the solvent system stationary phase (aqueous phase) was ca. 80%. The system with the acid and ethanol volume gradient consisting of BuOH-EtOH-NaClsolution-H2O-H3PO4 (v/v/v/v/v, 1300:200-240:1300:700:2.5-4.5) pumped at 2.0 ml/min was the most effective for a separation of betanin/isobetanin, 17-decarboxy-betanin/-isobetanin, 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin pairs as well as neobetanin. The pigments were detected by LC-DAD and LC-MS. The results are crucial in the application of completely food-grade solvent systems in separation of food-grade compounds as well, and the systems can possibly be extended to other ionizable and polar compounds with potential health benefits. In particular, the method is applicable for the isolation and purification of betalains present in such rich sources as B. vulgaris L. roots as well as cacti fruits and Amaranthaceae flowering plants due to modification possibilities of the solvent systems polarity. PMID:25595533

  15. Experimental and mathematical modeling studies of the separation of zinc blende and wurtzite phases of CdS nanorods by density gradient ultracentrifugation.

    PubMed

    Ma, Xiuju; Kuang, Yun; Bai, Lu; Chang, Zheng; Wang, Feng; Sun, Xiaoming; Evans, David G

    2011-04-26

    Identifying the phase purity of CdS nanorods (NRs) is complicated by the serious overlap between the X-ray diffraction peaks of zinc blende and wurtzite phases as well as anisotropic growth, which might hide a mixed phase. Here we show that the density gradient ultracentrifugation rate separation method can be used to sort CdS NRs synthesized under nitrogen according to differences in particle size and morphology. Furthermore, it was found that the different sized NRs formed in a single batch synthesis had different phases: the thinner ones (<3.5 nm in diameter) were predominantly wurtzite phase, while the thicker ones (>5 nm in diameter) were mainly zinc blende phase. Dark-field transmission electron microscopy (TEM) and high-resolution TEM images indicated the presence of numerous stacking faults in the thick zinc blende rods, while the wurtzite thin rods were exclusively single crystals. As a result of the differences in phase and stacking faults, the NRs showed different photoluminescent properties. The development of an effective way of separating such NRs thus leads to further insight into the differences in phase, structure, and optical properties between individual colloidal particles synthesized in a single batch. A preliminary mathematical model of the separation process has been proposed.

  16. A reproducible and high-throughput HPLC/MS method to separate sarcosine from α- and β-alanine and to quantify sarcosine in human serum and urine.

    PubMed

    Meyer, Tamra E; Fox, Stephen D; Issaq, Haleem J; Xu, Xia; Chu, Lisa W; Veenstra, Timothy D; Hsing, Ann W

    2011-07-15

    While sarcosine was recently identified as a potential urine biomarker for prostate cancer, further studies have cast doubt on its utility to diagnose this condition. The inconsistent results may be due to the fact that alanine and sarcosine coelute on an HPLC reversed-phase column and the mass spectrometer cannot differentiate between the two isomers, since the same parent/product ions are generally used to measure them. In this study, we developed a high-throughput liquid chromatography-mass spectrometry (LC-MS) method that resolves sarcosine from alanine isomers, allowing its accurate quantification in human serum and urine. Assay reproducibility was determined using the coefficient of variation (CV) and intraclass correlation coefficient (ICC) in serum aliquots from 10 subjects and urine aliquots from 20 subjects across multiple analytic runs. Paired serum/urine samples from 42 subjects were used to evaluate sarcosine serum/urine correlation. Both urine and serum assays gave high sensitivity (limit of quantitation of 5 ng/mL) and reproducibility (serum assay, intra- and interassay CVs < 3% and ICCs > 99%; urine assay, intra-assay CV = 7.7% and ICC = 98.2% and interassay CV = 12.3% and ICC = 94.2%). In conclusion, this high-throughput LC-MS method is able to resolve sarcosine from α- and β-alanine and is useful for quantifying sarcosine in serum and urine samples.

  17. 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.

  18. Effect of salt concentration gradient on separation of different types of specific immunoglobulins by ion-exchange chromatography on DEAE cellulose.

    PubMed

    Tishchenko, G A; Bleha, M; Skvor, J; Bostík, T

    1998-02-27

    A three-stage process, consisting of an ammonium sulfate precipitation step, dialysis desalination with microporous anion-exchange Neosepta membranes and anion-exchange chromatography on DEAE-cellulose DE-52 was used for the isolation of mouse monoclonal antibodies specific against different antigens. The ascites fluids contained monoclonal antibodies against human IgG, against horseradish peroxidase and against the heavy chain of human IgM. The effect of the salt concentration gradient in the elution buffer was examined with the aim of optimizing chromatographic conditions. The quality of separation of protein zones was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The immunoreactivity of purified monoclonal antibodies was determined by enzyme-linked immunosorbent assay using a solid-phase adsorbed antigens against which each monoclonal antibody type was directed.

  19. Enrichment of the glycoalkaloids alpha-solanine and alpha-chaconine from potato juice by adsorptive bubble separation using a pH gradient.

    PubMed

    Backleh, Marlène; Ekici, Perihan; Leupold, Günther; Coelhan, Mehmet; Parlar, Harun

    2004-08-01

    For the first time, the solanidine alkaloids alpha-solanine and alpha-chaconine could be quantitatively enriched from potato juice by Adsorptive Bubble Separation (ABS) with a pH gradient. The enrichment into the foam was influenced by the pH value, bubble size, and gas flow rate. The efficiency was highest on using diluted samples with a concentration between 2 and 6 mg L(-1) of the alkaloids at pH 6.0. The experiments with a standard solution of each alkaloid confirmed that these substances can be quantitatively enriched into the 'spumat' without surface active potato proteins. The transfer into the foam fraction under these conditions was similar to that from the aqueous potato extract.

  20. 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

  1. 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.

  2. Simultaneous separation and identification of oligomeric procyanidins and anthocyanin-derived pigments in raw red wine by HPLC-UV-ESI-MSn.

    PubMed

    Pati, S; Losito, I; Gambacorta, G; La Notte, E; Palmisano, F; Zambonin, P G

    2006-07-01

    Samples of raw red wine (Primitivo di Manduria, Apulia, Southern Italy) were analysed without any pre-treatment (except 1:2 dilution with water) using HPLC with detection based on UV absorbance and Electrospray Ionisation Sequential Mass Spectrometry (ESI-MSn, with n = 1-3) in a series configuration. In particular, absorbance at 520 nm was monitored for UV detection in order to identify pigments responsible for wine colour. On the other hand, two subsequent stages of MS detection based on positive ions were adopted. The first consisted of an explorative MS acquisition, aimed at the individuation of the m/z ratios for positively charged compounds; the second was based on fragmentation of the detected ions within an ion trap analyser, followed by MS/MS and, if required, MS3 acquisitions. The synergy between UV detection and MSn analysis led to the identification of 41 pigments, which can be classified into five groups: grape anthocyanins, pyranoanthocyanins, vinyl-linked anthocyanin-flavanol pigments, ethyl-bridged anthocyanin-flavanol pigments and flavanol-anthocyanin compounds. Many isomeric and oligomeric structures were found within each group. A further class of compounds, not absorbing in the visible spectrum, could be also characterised by ESI-MSn and corresponded to B-type procyanidins, i.e. proanthocyanidins arising from C4-->C8/C4-->C6 couplings between catechin or epicatechin units. In particular, oligomeric structures (from dimers to pentamers), often present with several isomers, were identified and their fragmentation patterns clarified. PMID:16770836

  3. 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...

  4. 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

  5. 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.

  6. 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.

    PubMed

    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.

  7. Chiral separation of cathinone and amphetamine derivatives by HPLC/UV using sulfated ß-cyclodextrin as chiral mobile phase additive.

    PubMed

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

    2014-08-01

    In the last years the identification of new legal and illegal highs has become a huge challenge for the police and prosecution authorities. In an analytical context, only a few analytical methods are available to identify these new substances. Moreover, many of these recreational drugs are chiral and it is supposed that the enantiomers differ in their pharmacological potency. Since nonenantioselective synthesis is easier and cheaper, they are mainly sold as racemic mixtures. The goal of this research work was to develop an inexpensive method for the chiral separation of cathinones and amphetamines. This should help to discover if the substances are sold as racemic mixtures and give further information about their quality as well as their origin. Chiral separation of a set of 6 amphetamine and 25 cathinone derivatives, mainly purchased from various Internet shops, is presented. A LiChrospher 100 RP-18e, 250 x 4 mm, 5 µm served as the stationary phase. The chiral mobile phase consisted of methanol, water, and sulfated ß-cyclodextrin. Measurements were performed under isocratic conditions in reversed phase mode using UV detection. Four model compounds of the two substance classes were used to optimize the mobile phase. Under final conditions (methanol:water 2.5:97.5 + 2% sulfated ß-cyclodextrin) enantiomers of amphetamine and five derivatives were baseline separated within 23 min. In all, 17 cathinones were completely or partially chirally separated. However, as only 3 of 25 cathinones were baseline resolved, the application of this method is limited for cathinone analogs. Additionally, the results were compared with an RP-8e column.

  8. 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.

  9. 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.

  10. [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

  11. Application of an efficient strategy based on MAE, HPLC-DAD-MS/MS and HSCCC for the rapid extraction, identification, separation and purification of flavonoids from Fructus Aurantii Immaturus.

    PubMed

    Wang, Chen; Pan, Yaju; Fan, Guorong; Chai, Yifeng; Wu, Yutian

    2010-03-01

    This study presents an efficient strategy based on microwave-assisted extraction (MAE), HPLC-DAD-MS/MS and high-speed counter-current chromatography (HSCCC) for the rapid extraction, identification, separation and purification of active components from the traditional Chinese medicine Fructus Aurantii Immaturus. An LC-DAD-MS/MS method was applied for the screening and structural identification of main components in crude extract, and five components were preliminarily identified as neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin according to their UV and mass spectra. An efficient MAE method for the extraction of the three most abundant components (narirutin, naringin and neohesperidin) was optimized by the combination of univariate and multivariate approaches. The crude extract was then separated and purified by HSCCC and a total of 61.6 mg of narirutin, 207.3 mg of naringin and 159.5 mg of neohesperidin at high purities of 98.1, 97.2 and 99.5%, respectively, were obtained from 1.42 g of crude extract. The recoveries of these compounds were 86, 93 and 89%, respectively.

  12. Precolumn o-phthalaldehyde-N-acetyl-L-cysteine derivatization followed by RP-HPLC separation and fluorescence detection of sitagliptin enantiomers in rat plasma.

    PubMed

    Nageswara Rao, R; Sravan, B; Ramakrishna, K; Saida, Shaik; Padiya, Raju

    2013-12-01

    An indirect reversed-phase high-performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o-phthalaldehyde and N-acetyl-L-cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP-18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50-5000 ng/ mL for both enantiomers. The intra- and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments.

  13. Development and validation of a reversed-phase HPLC method for separation and simultaneous determination of process-related substances of mirtazapine in bulk drugs and formulations.

    PubMed

    Rao, R Nageswara; Raju, A Narasa

    2009-03-01

    A simple and rapid reversed-phase high-performance liquid chromatographic method has been developed for the separation and simultaneous determination of related substances of mirtazapine in bulk drugs and pharmaceutical formulations. Six impurities, including one degradation product of mirtazapine, have been separated on a BDS Hypersil (4.6 x 250 mm; particle size 5 microm) column with a mobile phase consisting of 0.3% triethylamine (pH 3.0)-acetonitrile (78:22 v/v) eluted in an isocratic mode and monitored with a photo diode array detector at 215 nm. The chromatographic behavior of all the analytes was studied under variable compositions of different solvent systems, temperatures, buffer concentrations, and pH values. The method was validated in terms of accuracy, precision, and linearity. The inter- and intra-day assay precision was found to be < 0.98% [relative standard deviation; (RSD)] and the recoveries were in the range 95.54-102.22% with RSD < 2.21%. The correlation coefficients for calibration curves for mirtazapine as well as impurities were in the range of 0.9941-0.9999. The method was successfully applied to the analysis of commercial formulations and the recoveries of mirtazapine were in the range of 99.38-100.73% with < 0.52% RSD. The method is useful not only for rapid evaluation of the purity of mirtazapine, but also for the simultaneous determination of related substances in bulk drugs and pharmaceutical formulations.

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

    PubMed

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

    2010-10-27

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

  15. 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.

  16. 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

  17. Separation of dansylated 17β-estradiol, 17α-estradiol, and estrone on a single HPLC column for simultaneous quantitation by LC-MS/MS.

    PubMed

    Szarka, Szabolcs; Nguyen, Vien; Prokai, Laszlo; Prokai-Tatrai, Katalin

    2013-04-01

    We show here that baseline separation of dansylated estrone, 17β-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs (13)C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®. PMID:23371528

  18. Separation of dansylated 17β-estradiol, 17α-estradiol and estrone on a single HPLC column for simultaneous quantitation by LC-MS/MS

    PubMed Central

    Szarka, Szabolcs; Nguyen, Vien; Prokai, Laszlo; Prokai-Tatrai, Katalin

    2013-01-01

    We show here that baseline separation of dansylated estrone, 17β-estradiol and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope-dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs 13C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®. PMID:23371528

  19. Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high-gradient magnetic separation.

    PubMed

    Müller, Christine; Heidenreich, Elena; Franzreb, Matthias; Frankenfeld, Katrin

    2015-01-01

    Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed-bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed 'rotor-stator' type High Gradient Magnetic Fishing (HGMF) system for large-scale application, currently up to 100 g of magnetic adsorbents. Initially, the separation process design was optimized for binding and elution conditions for the target protein in mL scale. Subsequently, the magnetic filter for particle separation was characterized. Based on these results, a purification process for eCG was designed consisting of (i) pretreatment of the horse serum; (ii) binding of the target protein to magnetic ion exchange adsorbents in a batch reactor; (iii) recovery of loaded functionalized adsorbents from the pretreated solution using HGMF; (iv) washing of loaded adsorbents to remove unbound proteins; (v) elution of the target protein. Finally, the complete HGMF process was automated and conducted with either multiple single-cycles or multicycle operation of four sequential cycles, using batches of pretreated serum of up to 20 L. eCG purification with yields of approximately 53% from single HGMF cycles and up to 80% from multicycle experiments were reached, with purification and concentration factors of around 2,500 and 6.7, respectively.

  20. Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC-ESI-MS/MS Analysis.

    PubMed

    Lin, Chia-Wei; Haeuptle, Micha A; Aebi, Markus

    2016-09-01

    Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides. PMID:27487254

  1. Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC-ESI-MS/MS Analysis.

    PubMed

    Lin, Chia-Wei; Haeuptle, Micha A; Aebi, Markus

    2016-09-01

    Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides.

  2. 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.

  3. 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.

  4. Qualitative and quantitative analysis of cinobufacini injection using rapid separation liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and HPLC-photodiode array detection, a feasible strategy for the quality control of Chinese medicine injections.

    PubMed

    Zhao, Haiyu; Wu, Xu; Wang, Hongjie; Gao, Bo; Yang, Jian; Si, Nan; Bian, Baolin

    2013-02-01

    Cinobufacini injection, prepared from the skin of Bufo bufo gargarizans Cantor, has presented its significant effects on the treatment of hepatitis and various cancers in the clinic. However, as an unclear complex chemical system, the optimization of its quality control markers has been a long-term challenge. In present study, a feasible strategy integrated markers screening, determination, and statistical analysis was efficiently proposed, especially for the undefined Chinese medicine injections. First, rapid separation LC-quadrupole-TOF-MS method was applied in the identification of 19 major compounds in the cinobufacini injection for the first time. Further, nine high-level contents active compounds were selected as quality control markers for the quantification analysis. An acceptable and validated determination method was established in 17 batches of cinobufacini injection by HPLC-photodiode array detection method, including linear regression relationship (r(2), 0.9996-1), precisions (RSD, 0.02-1.35%), repeatability (RSD, 0.05-1.97%), stability (RSD, 0.1-3.85%), and recovery (95.88-104.89%). Each analyte was detected at its maximum ultraviolet spectra wavelength. Finally, based on the quantification results, principal component analysis was performed on the quality assessment of cinobufacini injections. This three-step strategy provides a newly feasible solution for the quality control of Chinese medicine injections.

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

    PubMed

    Jung, Stephanie; Effelsberg, Uwe; Tallarek, Ulrich

    2011-12-01

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

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

    PubMed

    Jung, Stephanie; Effelsberg, Uwe; Tallarek, Ulrich

    2011-12-01

    Dynamic changes in mobile phase composition during high-performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray modes. We investigate the impact of the eluent composition on spray stability and MS response by infusion and injection experiments with a small tetrapeptide in water-acetonitrile mixtures. The employed HPLC/electrospray (ESI)-MS configuration uses a microchip equipped with an enrichment column, a separation column, and a makeup flow (MUF) channel. One nano pump is connected to the separation column, while a second one delivers solvent of exactly inverted composition to the MUF channel. Both solvent streams are united behind the separation column, before the ESI tip, such that the resulting electrosprayed solution always has identical composition during a gradient elution. Analyte peak parameters without and with MUF compensation are determined and discussed with respect to the electrospray mode and eluent composition. The postcolumn MUF significantly improves spray and signal stability over the entire solvent gradient, without compromising the performance of the HPLC separation column. It can also be conveniently implemented on microchip platforms. PMID:22022890

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. Validation of a HPLC method for simultaneous determination of five sunscreens in lotion preparation.

    PubMed

    Kedor-Hackmann, E R M; De Lourdes Pérez González, M L; Singh, A K; Santoro, M I R M

    2006-06-01

    The aim of this research was to develop and validate a high-performance liquid chromatographic (HPLC) method for simultaneous determination of five sunscreens, namely benzophenone-3 (B-3), butyl methoxydibenzoylmethane (BM), octyl methoxycinnamate (OM), octyl salicylate (OS) and homosalate (HS). The separation and quantitative determination was made by HPLC at 40 +/-1 degrees C with a gradient elution from 10% to 100% mobile phase B in mobile phase A. The gradient liquid chromatographic system constituted of mobile phase A [acetonitrile : water (10 : 90 v/v)] and mobile phase B [acetonitrile : water (90 : 10 v/v)], at a flow rate of 1.0 mL min(-1) and ultraviolet detection at 310 nm. The separation was obtained with two Waters reversed phase columns: Novapack C-18 and Symmetry((R)) C-18 connected in series. All sunscreens were efficiently separated within 17 min. The coefficient of correlation and average recovery for B-3, BM, OM, OS and HS were 0.9798 and 98.5%, 0.9672 and 98.8%, 0.9922 and 99.1%, 0.9961 and 98.9% and 0.9909 and 99.4% respectively. The relative standard deviations obtained were between 1.07% and 2.44%. The excipients did not interfere in the analysis. The results showed that the proposed method could be used for rapid and simultaneous determination of B-3, BM, OM, OS and HS in sunscreen lotions with precision, accuracy and specificity.

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

    PubMed

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

    2009-10-01

    The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD-HPLC-ESI-TOF-MS and IT-MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF-MS in order to obtain molecular formula and exact mass. Posterior analyses with IT-MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode. PMID:19750503

  13. Separation and purification of α-glucosidase inhibitors from Polygonatum odoratum by stepwise high-speed counter-current chromatography combined with Sephadex LH-20 chromatography target-guided by ultrafiltration-HPLC screening.

    PubMed

    Zhou, Xiaoling; Liang, Junsheng; Zhang, Yi; Zhao, Huading; Guo, Ying; Shi, Shuyun

    2015-03-15

    Although Polygonatum odoratum has been widely used as medicinal plant and food supplement for treating diabetes, little is known regarding its bioactive components. In this study, ultrafiltration-HPLC based ligand screening was developed to screen α-glucosidase inhibitors from P. odoratum for the first time. Then bioactive components were target-guided separated by combining stepwise high-speed counter-current chromatography (HSCCC) using petroleum ether-ethyl acetate-methanol-water (1:4:0.8:4.2, v/v/v/v), (1:4:1.8:3.2, v/v/v/v) and (1:4:2.3:2.7, v/v/v/v) as solvent systems with Sephadex LH-20 chromatography eluted by MeCN-MeOH (1:1, v/v). Five phenethyl cinnamides, N-cis-feruloyloctopamine (1); N-trans-p-coumaroyloctopamine (2), N-trans-feruloyloctopamine (3), N-trans-p-coumaroyltyramine (4) and N-trans-feruloyltyramine (5), and four homoisoflavanones, (3R)-5,7-dihydroxyl-3-(2',4'-dihydroxylbenzyl)-chroman-4-one (6), (3R)-5,7-dihydroxyl-6-methyl-3-(4'-hydroxylbenzyl)-chroman-4-one (7), (3R)-5,7-dihydroxyl-6-methyl-8-methoxyl-3-(4'-hydroxylbenzyl)-chroman-4-one (8); and (3R)-5,7-dihydroxyl-6,8-dimethyl-3-(4'-hydroxylbenzyl)-chroman-4-one) (9), with purity over 98.5% were purified, and their structures were identified by UV, MS, and (1)H NMR. Notably, compounds 2 and 4 were first reported in genus Polygonatum, while compound 1 was first obtained from family Liliaceae. In addition, α-glucosidase inhibitory activities of compounds 1-9 were evaluated, and compounds 2 and 4 exhibited stronger α-glucosidase inhibitory activity with IC50 values of 2.3 and 2.7μM. The results suggested the potential medicinal use of P. odoratum, and the technology could be widely applied for rapid screening and preparative separation of a group of bioactive compounds from complex matrix.

  14. Simultaneous Fe, S and P Speciation Using HPLC=HR-ICPMS

    NASA Astrophysics Data System (ADS)

    Hamester, M.; Lindemann, T.; Rottmann, L.; Douthitt, C. B.

    2004-12-01

    Iron, sulphur and phosphorus-containing biomolecules play important roles in biochemistry and proteomics and are part of the metallome of an organism. Analysis of HPLC peaks by ICPMS for these metals is a difficult analytical challenge because of polyatomic interferences: carbon, oxygen and nitrogen based interferences (NO+, NOH+, COH+, H3CO+, O2+) are the principal limitation in P and S detection and ArO+ is the principal interference on Fe. Polyatomic interferences formed by the elements in the different mobile phases can be separated from P and S in the molecules of interest, allowing the use of gradient elution. With high resolution ICPMS (HR-ICPMS), P and S can be completely resolved from these interferences even in 100 % acetonitrile. Protein phosphorylation can be determined accurately by HR-ICPMS, either on HPLC peaks or by laser ablation of gel electrophoresis spots. The high power magnet field regulator of the Finnigan ELEMENT2 enables rapid mass scanning, e.g. a duty cycle of 99% is obtained for the simultaneous detection of P and S. In order to show the potential of HR-ICPMS for metallomics, mixtures of deoxyribonucleotides (dAMP, dTMP, dGMP, dCMP) and peptides have been separated by HPLC with simultaneous on-line quantitation of P and S, and hemoglobin and myoglobin were analyzed for Fe/S ratios and quantitative determination of Fe.

  15. 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

  16. 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.

  17. 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.

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

    PubMed

    Downey, Mark O; Rochfort, Simone

    2008-08-01

    A limitation of large-scale viticultural trials is the time and cost of comprehensive compositional analysis of the fruit by high-performance liquid chromatography (HPLC). In addition, separate methods have generally been required to identify and quantify different classes of metabolites. To address these shortcomings a reversed-phase HPLC method was developed to simultaneously separate the anthocyanins and flavonols present in grape skins. The method employs a methanol and water gradient acidified with 10% formic acid with a run-time of 48 min including re-equilibration. Identity of anthocyanins and flavonols in Shiraz (Vitis vinifera L.) skin was confirmed by mass spectral analysis. PMID:18573501

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

    PubMed

    Šesták, Jozef; Kahle, Vladislav

    2014-07-11

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

  20. 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

  1. 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

  2. Quantitative analysis combined with chromatographic fingerprint for comprehensive evaluation of Xiaoer Chaigui Tuire granules by HPLC-DAD.

    PubMed

    Liu, Hong-Ming; Nie, Lei

    2015-01-01

    Quantitative analysis of eight major components combined with chromatographic fingerprint based on high performance liquid chromatography coupled with diode array detector (HPLC-DAD) was developed for the quality evaluation of Xiaoer Chaigui Tuire granules (XCTG), a traditional Chinese medicine (TCM) preparation. Each compound was analyzed by comparing its retention time and UV spectrum of each chromatographic peak with the corresponding retention time and UV spectrum of each standard compound. Baseline separation was achieved on an Agilent Zorbax SB-C18 column with gradient elution of acetonitrile and 0.1% (v/v) phosphoric acid. The developed method was validated by linearity, precision, repeatability, stability and recovery and was subsequently applied to quality evaluation of 12 batches of XCTG with similarity analysis, principal component analysis and cluster analysis. Quantitative analysis combined with HPLC fingerprint could offer an efficient, reliable and practical approach for quality evaluation of XCTG.

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

    PubMed

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

    2012-09-01

    This paper describes the determination of fatty acid composition of coffee, citrus and rum distillery wastes using reversed-phase high-performance liquid chromatography (RP-HPLC). Lipid extracts of the waste samples are derivatized with phenacyl bromide and their phenacyl esters are separated on a C8 reversed-phase column by using continuous gradient elution with water and acetonitrile. The presence of saturated and unsaturated fatty acids in quantifiable amounts in the examined wastes, as well as the high percentage recoveries, are clear indications that these wastes have potential value as inexpensive sources of lipids. The HPLC procedures described here could be adopted for further analysis of materials of this nature. PMID:22595260

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

    PubMed

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

    2014-09-24

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

  5. 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.

  6. An HPLC chromatographic reactor approach for investigating the hydrolytic stability of a pharmaceutical compound.

    PubMed

    Skrdla, Peter J; Abrahim, Ahmed; Wu, Yan

    2006-06-01

    The solution-phase hydrolysis kinetics of the Aprepitant (Emend) prodrug, Fosaprepitant Dimeglumine, were investigated using an HPLC chromatographic reactor approach. The term 'chromatographic reactor' refers to the use of an analytical-scale column as both a flow-through reactor and, simultaneously, as separation medium for the reactant(s) and product(s). Recently, we reported a novel mathematical treatment for the kinetic data obtained from chromatographic reactors, which we believe is superior to other treatments in terms of its accuracy, robustness and ease of implementation. In this work, we demonstrate that our treatment may be applied equally well to HPLC reactors, as previously we studied only GC reactors. It is found that the hydrolysis of Fosaprepitant Dimeglumine (FD) has an apparent activation energy of 107 kJ/mol when the reaction is investigated on-column, using the gradient elution conditions of the validated HPLC impurity profile method for this compound. For comparison, the activation energy determined for the same reaction occurring in a quiescent solution consisting of a fixed ratio of acetonitrile-0.1% v/v aqueous H3PO4 (50:50, v/v) is 91 kJ/mol, calculated using direct application of the Arrhenius equation. The data presented show that, when used as a screening tool, chromatographic reactors may be feasible for use in the pharmaceutical industry to quickly gauge the relative stabilities of various compounds with similar degradation pathways. PMID:16524680

  7. [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

  8. Performance evaluation of an aqueous-organic phase separator for post-column reactions in high-performance liquid chromatography, and its application to the enhanced detection of some basic drugs of abuse.

    PubMed

    Roy, I M; Jefferies, T M

    1990-01-01

    A phase separator is described that is suitable for post-column HPLC applications. It operates with commonly used HPLC eluents and immiscible organic solvents as long as the two phases remain immiscible. It is compatible with gradient elution systems. Separation efficiency is routinely better than 0.8, which ensures that analyte peak heights are about 95% of the maximum height under these conditions. An application for the detection of pethidine, cocaine, methadone, piritramide and dipipanone at 0.8-1.8 ng on-column loadings is described.

  9. 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

  10. Near-wall scaling for turbulent boundary layers with adverse pressure gradient. A priori tests on DNS of channel flow with periodic hill constrictions and DNS of separating boundary layer

    NASA Astrophysics Data System (ADS)

    Manhart, Michael; Peller, Nikolaus; Brun, Christophe

    2008-05-01

    A new extended inner scaling is proposed for the wall layer of wall-bounded flows under the influence of both wall shear stress and streamwise pressure gradient. This scaling avoids problems of the classical wall coordinates close to flow separation and reattachment. Based on the proposed extended velocity and length scales a universal nondimensional family of velocity profiles is derived for the viscous region in the vicinity of a wall that depend on wall distance and a parameter α quantifying the importance of the streamwise pressure gradient with respect to the wall shear stress in the momentum balance. The performance of the proposed extended scaling is investigated in two different flow fields, a separating and reattaching turbulent boundary layer and a turbulent flow over a periodic arrangement of smoothly contoured hills. Both flows are results of highly resolved direct numerical simulation (DNS). The results show that the viscous assumptions are valid up to about two extended wall units. If the profiles are scaled by the extended inner coordinates, they seem to behave in a universal way. This gives rise to the hope that a universal behavior of velocity profiles can be found in the proposed extended inner coordinates even beyond the validity of the extended viscous law of the wall.

  11. 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.

  12. 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.

  13. [Erythrocyte removal from bone marrow by density gradient separation using the COBE 2991 cell processor with the triple-bag processing set].

    PubMed

    Cappellesso-Fleury, S; Rage, C; Tschaggeny, F; Gaudé, J; Gomez, M; Bourin, P

    2009-03-01

    ABO-incompatible bone marrow transplantation requires red blood cell depletion. Lots of laboratory adopted the technique of density gradient centrifugation (Ficoll-hypaque) using the COBE 2991 cell processor with simple-bag processing set. However, tubing of this set is not adapted to the currently available peristaltic pumps. Moreover, two other sets are required: one for the buffy-coat and one for postgradient cell washing. We developed a method using triple-bag processing set to conduct whole-step procedure (concentration, Ficoll and washing). Peristaltic PVC tubing is provided in one line of the set allowing a safe processing without several connections thus reducing risks of microbial contamination. First, we used buffy-coat of total blood for training, then, we carried out red cell depletion of healthy bone marrow donors. The red blood cell depletion was 97.9+/-1.1% and CD34+ recovery was 89.6+/-8.7%. These results are very close to those obtained with the simple-bag set (red cell depletion.=94.0+/-6.8% and CD34+ recovery=95.9+/-20.3%). We conclude that the triple-bag system, very little used in France, is practical, simplified the manipulation and is more safety than the simple-bag set.

  14. HPLC and chemometric methods for the simultaneous determination of cyproheptadine hydrochloride, multivitamins, and sorbic acid.

    PubMed

    el-Gindy, Alaa; el-Yazby, Fawzy; Mostafa, Ahmed; Maher, Moustafa M

    2004-06-29

    Three methods are presented for the simultaneous determination of cyproheptadine hydrochloride (CP), thiamine hydrochloride (B1), riboflavin-5-phosphate sodium dihydrate (B2), nicotinamide (B3), pyridoxine hydrochloride (B6), and sorbic acid (SO). The chromatographic method depends on a high performance liquid chromatographic (HPLC) separation on a reversed-phase, RP 18 column. Elution was carried out with 0.1% methanolic hexane sulphonic acid sodium salt (solvent A) and 0.01 M phosphate buffer containing 0.1% hexane sulphonic acid sodium salt, adjusted to an apparent pH of 2.7 (solvent B). Gradient HPLC was used with the solvent ratio changed from 20:80 to 70:30 (over 9 min), then to 80:20 (over 11 min) for solvent A:B, respectively. Quantitation was achieved with UV detection at 220 and 288 nm based on peak area. The other two chemometric methods applied were principal component regression (PCR) and partial least squares (PLS). These approaches were successfully applied to quantify each drug in the mixture using the information included in the UV absorption spectra of appropriate solutions in the range 250-290 nm with the intervals Deltalambda = 0.4 nm at 100 wavelengths. The chemometric methods do not require any separation step. The three methods were successfully applied to a pharmaceutical formulation and the results were compared with each other.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. Gradient networks

    NASA Astrophysics Data System (ADS)

    Toroczkai, Zoltán; Kozma, Balázs; Bassler, Kevin E.; Hengartner, N. W.; Korniss, G.

    2008-04-01

    Gradient networks are defined (Toroczkai and Bassler 2004 Nature 428 716) as directed graphs formed by local gradients of a scalar field distributed on the nodes of a substrate network G. We present the derivation for some of the general properties of gradient graphs and give an exact expression for the in-degree distribution R(l) of the gradient network when the substrate is a binomial (Erd{\\;\\kern -0.10em \\raise -0.35ex \\{{^{^{\\prime\\prime}}}}\\kern -0.57em \\o} s-Rényi) random graph, G_{N,p} , and the scalars are independent identically distributed (i.i.d.) random variables. We show that in the limit N \\to \\infty, p \\to 0, z = pN = \\mbox{const} \\gg 1, R(l)\\propto l^{-1} for l < l_c = z , i.e., gradient networks become scale-free graphs up to a cut-off degree. This paper presents the detailed derivation of the results announced in Toroczkai and Bassler (2004 Nature 428 716).

  1. Separation and identification of phenolic compounds of extra virgin olive oil from Olea europaea L. by HPLC-DAD-SPE-NMR/MS. Identification of a new diastereoisomer of the aldehydic form of oleuropein aglycone.

    PubMed

    Pérez-Trujillo, Míriam; Gómez-Caravaca, Ana María; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto; Parella, Teodor

    2010-08-25

    The phenolic fraction of a monovarietal extra virgin olive oil (EVOO) from Olea europaea L. var. Cornezuelo was studied by the hyphenated HPLC-DAD-SPE-NMR/MS techniques. This survey led to the identification of 25 main compounds. One was identified as a new diastereoisomer of the aldehydic form of oleuropein aglycone (AOA) and characterized by 1D and 2D NMR techniques. The relative configuration of this new AOA was determined as 5R*,8S*,9S* on the basis of the results obtained from the combination of NOE experiments and Monte Carlo conformational search calculations. Assuming, as for the described diastereoisomers, that the new AOA comes from the natural oleuropein aglycone (OA), the absolute configuration was proposed as 5S,8R,9R.

  2. 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

  3. Determination of active substances in binary mixture antiparasitic veterinary formulations by HPLC.

    PubMed

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

    2011-01-01

    The purpose of the study was to develop a simple, versatile HPLC method for the identification and quantification of praziquantel and ivermectin (in Equimax) or praziquantel and abamectin (in Abamitel Plus). A satisfactory separation was obtained using the Supelcosil LC-ABZ+ column in gradient system with a mobile phase A: acetonitrile / water in 40:60 ratio and phase B: acetonitrile. The UV detection was set at 245 nm. The correlation coefficient values (> or = 0,998) for all active substances confirmed that the calibration curves (peak area vs. concentration) are linear. The results of the quantification and the statistical evaluation confirmed that the method is accurate and precise. It can also be applied to confirm the identity of benzyl alcohol, methyl p-hydroxybenzoate and propyl p-hydroxybenzoate in Abamitel Plus formulation. PMID:21796928

  4. 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.

  5. Quality evaluation of Carthami Flos by HPLC-UV.

    PubMed

    Li, Ying; Piao, Donggen; Zhang, Haiyan; Kim, Taein; Lee, Seung-Ho; Chang, Hyeun Wook; Woo, Mi-Hee; Son, Jong Keun

    2015-01-01

    A HPLC-DAD method was developed for simultaneous determination of four marker compounds, kaempferol-3-O-rutinoside, safflomin A, safflomin B and bidenoside C, in the extract of the flowers of Carthamus tinctorius Linne. Natural samples were extracted in 50% aqueous methanol by ultra-sonication for 60 min. Marker compounds were separated on a C18 column by two-step gradient elution of mobile phase (acetonitrile/water) at a flow rate of 0.75 mL/min and detected at 210 nm. The retention times of safflomin A and safflomin B were shifted according to the pH of the mobile phase. The optimized analytical method was validated to confirm linearity, precision, accuracy and stability of marker compounds. The method was successfully employed to analyze 17 natural samples from different regions, and the data matrix was monitored and visualized by principal component analysis. The assay method could be applied for quality control of the flowers of C. tinctorius Linne.

  6. 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

  7. Combination of TREF, high-temperature HPLC, FTIR and HPer DSC for the comprehensive analysis of complex polypropylene copolymers.

    PubMed

    Cheruthazhekatt, Sadiqali; Pijpers, Thijs F J; Mathot, Vincent B F; Pasch, Harald

    2013-11-01

    A novel, powerful analytical technique, preparative temperature rising elution fractionation (prep TREF)/high-temperature (HT)-HPLC/Fourier transform infrared spectroscopy (FTIR)/high-performance differential scanning calorimetry (HPer DSC)), has been introduced to study the correlation between the polymer chain microstructure and the thermal behaviour of various components in a complex impact polypropylene copolymer (IPC). For the comprehensive analysis of this complex material, in a first step, prep TREF is used to produce less complex but still heterogeneous fractions. These chemically heterogeneous fractions are completely separated by using a highly selective chromatographic separation method--high-temperature solvent gradient HPLC. The detailed structural and thermal analysis of the HPLC fractions was conducted by offline coupling of HT-HPLC with FTIR spectroscopy and a novel DSC method--HPer DSC. Three chemically different components were identified in the mid-elution temperature TREF fractions. For the first component, identified as isotactic polypropylene homopolymer by FTIR, the macromolecular chain length is found to be an important factor affecting the melting and crystallisation behaviour. The second component relates to ethylene-propylene copolymer molecules with varying ethylene monomer distributions and propylene tacticity distributions. For the polyethylene component (last eluting component in all semi-crystalline TREF fractions), it was found that branching produced defects in the long crystallisable ethylene sequences that affected the thermal properties. The different species exhibit distinctively different melting and crystallisation behaviour, as documented by HPer DSC. Using this novel approach of hyphenated techniques, the chain structure and melting and crystallisation behaviour of different components in a complex copolymer were investigated systematically.

  8. "Measure Your Gradient": a new way to measure gradients in high performance liquid chromatography by mass spectrometric or absorbance detection.

    PubMed

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

    2014-11-21

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

  9. Determination of flavonol glycosides in green tea, oolong tea and black tea by UHPLC compared to HPLC.

    PubMed

    Jiang, Heyuan; Engelhardt, Ulrich H; Thräne, Claudia; Maiwald, Beate; Stark, Janina

    2015-09-15

    An UHPLC method for the determination of flavonol glycosides (FOG) from green and oolong tea vs. black tea has been developed for the first time. Sample clean-up method by means of polyamide column chromatography was optimized with multiple-step elution. Using UHPLC and HPLC with gradient elution and photodiode array detection, eighteen FOG compounds were determined with the aid of electrospray tandem mass spectrometry. These FOG compounds were qualified on both UHPLC and HPLC, and this UHPLC method successfully separated rutin (quercetin-3-O-rutinoside) and K-grg (kaempferol-3-O-glucorhamnoglucoside) while conventional HPLC method did not. The total amounts of FOG compounds in the tea samples were 2.32-5.67g/kg dry weight (calculated as aglycones), and there is no significant difference for the total FOG content among green tea, oolong tea and black tea. However, kaempferol glycosides are more abundant in green teas, while oolong tea has more quercetin and myricetin glycosides. In black tea quercetin glycosides were most abundant.

  10. Gradient elution in capillary electrochromatography

    SciTech Connect

    Anex, D.; Rakestraw, D.J.; Yan, Chao; Dadoo, R.; Zare, R.N.

    1997-08-01

    In analogy to pressure-driven gradient techniques in high-performance liquid chromatography, a system has been developed for delivering electroosmotically-driven solvent gradients for capillary electrochromatography (CEC). Dynamic gradients with sub-mL/min flow rates are generated by merging two electroosmotic flows that are regulated by computer-controlled voltages. These flows are delivered by two fused-silica capillary arms attached to a T-connector, where they mix and then flow into a capillary column that has been electrokinetically packed with 3-mm reversed-phase particles. The inlet of one capillary arm is placed in a solution reservoir containing one mobile phase and the inlet of the other is placed in a second reservoir containing a second mobile phase. Two independent computer-controlled programmable high-voltage power supplies (0-50 kV)--one providing an increasing ramp and the other providing a decreasing ramp--are used to apply variable high-voltage potentials to the mobile phase reservoirs to regulate the electroosmotic flow in each arm. The ratio of the electroosmotic flow rates between the two arms is changed with time according to the computer-controlled voltages to deliver the required gradient profile to the separation column. Experiments were performed to confirm the composition of the mobile phase during a gradient run and to determine the change of the composition in response to the programmed voltage profile. To demonstrate the performance of electroosmotically-driven gradient elution in CEC, a mixture of 16 polycyclic aromatic hydrocarbons (PAHs) was separated in less than 90 minutes. This gradient technique is expected to be well-suited for generating not only solvent gradients in CEC, but also other types of gradients such as pH- and ionic-strength gradients in capillary electrokinetic separations and analyses.

  11. Improved separation of furocoumarins of essential oils by supercritical fluid chromatography.

    PubMed

    Desmortreux, C; Rothaupt, M; West, C; Lesellier, E

    2009-10-16

    Separation of furocoumarins has become of a great interest for cosmetic industry and human health, since the recent directive of the European Union. Furocoumarins are a class of compounds presenting varied substituents linked mainly in two positions to an identical skeleton made by a furan ring bonded to a coumarin nucleus (Psoralen). The substituents are mainly methoxy, or alkyl chains, which can contain double bonds, hydroxyl or epoxy groups. Due to the variety of compounds, and their subtle structure differences, their separation requires high-performance methods. Multi-gradient high-performance liquid chromatography (HPLC) and two-dimensional chromatography are usually applied. This paper describes a new approach, by using super/subcritical fluid chromatography (SFC), with a green mobile phase: CO(2)-ethanol. The choice of the stationary phase from varied types of phases, and the effects of numerous analytical parameters (flow rate, modifier percentage, temperature and outlet pressure) are studied, described and discussed, on the basis of the separation of a complex sample: lemon residue. From these studies, isocratic conditions are determined to obtain a satisfactory separation in 10 min. A two-dimensional analysis was also investigated, by performing first a class fractionation of compounds on an ethylpyridine (EP) phase, then by separating each class on a pentafluorophenyl phase (Discovery HS F5) with the selected isocratic mobile phase. A gradient elution is also studied to improve separation of some minor compounds. Structure of the eluted compounds was determined by comparison with standards, HPLC-DAD, HPLC-MS analysis, and NMR analysis of collected fractions. All these approaches allow relating structure of compounds to retention behaviour, which is unusual due to the selected pentafluorophenyl stationary phase. PMID:19747687

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

    PubMed

    Hussien, Emad M

    2014-09-01

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

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

    PubMed

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

    2013-06-19

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

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

    PubMed

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

    2013-06-19

    The root of burdock ( Arctium lappa L.) is a commonly used vegetable in Asia. Fructooligosaccharides (FOS) are usually considered as its main bioactive components. Thus, quantitative analysis of these components is very important for the quality control of burdock. In this study, an HPLC-ELSD and microwave-assisted extraction method was developed for the simultaneous determination of seven FOS with degrees of polymerization (DP) between 3 and 9, as well as fructose, glucose, and sucrose in burdock from different regions. The separation was performed on a Waters XBridge Amide column (4.6 × 250 mm i.d., 3.5 μm) with gradient elution. All calibration curves for investigated analytes showed good linear regression (r > 0.9990). Their LODs and LOQs were lower than 3.63 and 24.82 μg/mL, respectively. The recoveries ranged from 99.2 to 102.6%. The developed method was successfully applied to determination of ten sugars in burdock from different locations of Asia. The results showed that the contents of FOS in different samples of burdock collected at appropriate times were similar, and the developed HPLC-ELSD with microwave-assisted extraction method is helpful to control the quality of burdock. PMID:23745967

  15. Simultaneous determination of prenylflavonoid and hop bitter acid in beer lee by HPLC-DAD-MS.

    PubMed

    Kao, T H; Wu, G Y

    2013-11-15

    An HPLC-DAD-MS method with high accuracy and precision was developed for determination of prenylflavonoids and hop bitter acids in beer lee, a by-product from beer brewing process. Four prenylflavonoids and nine hop bitter acids can be simultaneously separated in 29 min using a Thermo HyPURITY C18 column in combination with diode array dectector and mass spectrometer with HPLC solvent gradient system of phosphoric acid aqueous solution at pH 1.6 and acetonitrile at a flow rate of 1.5 mL/min and detection wavelength at 314 nm. Beer lee is found to contain isoxanthohumol (36.2 μg/g), xanthohumol (29.6 μg/g), 8-prenylnaringenin (7.84 μg/g), 6-prenylnaringenin (19.2 μg/g), cohumulone (44.7 μg/g), humulone (123 μg/g), adhumulone (21.8 μg/g), colupulone (44.2 μg/g), lupulone (33.2 μg/g), and adlupulone (5.76 μg/g).

  16. 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

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

    PubMed

    Appala, Raju N; Chigurupati, Sridevi; Appala, Raju V V S S; Krishnan Selvarajan, Kesavanarayanan; Islam Mohammad, Jahidul

    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

  18. 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

  19. Combining Step Gradients and Linear Gradients in Density.

    PubMed

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

    2015-06-16

    Combining aqueous multiphase systems (AMPS) and magnetic levitation (MagLev) provides a method to produce hybrid gradients in apparent density. AMPS—solutions of different polymers, salts, or surfactants that spontaneously separate into immiscible but predominantly aqueous phases—offer thermodynamically stable steps in density that can be tuned by the concentration of solutes. MagLev—the levitation of diamagnetic objects in a paramagnetic fluid within a magnetic field gradient—can be arranged to provide a near-linear gradient in effective density where the height of a levitating object above the surface of the magnet corresponds to its density; the strength of the gradient in effective density can be tuned by the choice of paramagnetic salt and its concentrations and by the strength and gradient in the magnetic field. Including paramagnetic salts (e.g., MnSO4 or MnCl2) in AMPS, and placing them in a magnetic field gradient, enables their use as media for MagLev. The potential to create large steps in density with AMPS allows separations of objects across a range of densities. The gradients produced by MagLev provide resolution over a continuous range of densities. By combining these approaches, mixtures of objects with large differences in density can be separated and analyzed simultaneously. Using MagLev to add an effective gradient in density also enables tuning the range of densities captured at an interface of an AMPS by simply changing the position of the container in the magnetic field. Further, by creating AMPS in which phases have different concentrations of paramagnetic ions, the phases can provide different resolutions in density. These results suggest that combining steps in density with gradients in density can enable new classes of separations based on density. PMID:25978093

  20. 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)

  1. Flow field thermal gradient gas chromatography.

    PubMed

    Boeker, Peter; Leppert, Jan

    2015-09-01

    Negative temperature gradients along the gas chromatographic separation column can maximize the separation capabilities for gas chromatography by peak focusing and also lead to lower elution temperatures. Unfortunately, so far a smooth thermal gradient over a several meters long separation column could only be realized by costly and complicated manual setups. Here we describe a simple, yet flexible method for the generation of negative thermal gradients using standard and easily exchangeable separation columns. The measurements made with a first prototype reveal promising new properties of the optimized separation process. The negative thermal gradient and the superposition of temperature programming result in a quasi-parallel separation of components each moving simultaneously near their lowered specific equilibrium temperatures through the column. Therefore, this gradient separation process is better suited for thermally labile molecules such as explosives and natural or aroma components. High-temperature GC methods also benefit from reduced elution temperatures. Even for short columns very high peak capacities can be obtained. In addition, the gradient separation is particularly beneficial for very fast separations below 1 min overall retention time. Very fast measurements of explosives prove the benefits of using negative thermal gradients. The new concept can greatly reduce the cycle time of high-resolution gas chromatography and can be integrated into hyphenated or comprehensive gas chromatography setups.

  2. HPLC of flavanones and chalcones in different species and clones of Salix.

    PubMed

    Krauze-Baranowska, Mirosława; Pobłocka-Olech, Loretta; Głód, Daniel; Wiwart, Marian; Zieliński, Jerzy; Migas, Piotr

    2013-01-01

    The SPE-HPLC method was developed to determine an isosalipurposide (5) and its derivative, 6"-O-p-coumaroyl ester (6) in the bark of eight taxa (I-VIII) belonging to three species of the genus Salix and originating from a natural habitat or cultivated for pharmaceutical purposes. The chalcones were separated by HPLC under gradient elution with the concentration of ACN increasing from 20% to 50% in 0.1% aqueous H3PO, (tG 15 min). The content of both compounds was determined by an external standardization with the use of isoliquiritigenin (7) as a reference substance - a commercially available chalcone, and also isosalipurposide (5) and its derivative, 6"-O-p-coumaroyl ester (6). The latter compound was isolated from the bark of Salix daphnoides (IV) by a CC and semi-preparative HPLC and its structure was elucidated by MS and NMR spectra. It was stated that 6"-O-p-coumaroylisosalipurposide (6), in addition to isosalipurposide (5), is a characteristic flavonoid for the S. daphnoides species. Moreover, the presence of these two chalcones was confirmed in the bark of S. acutifolia (I). Differences were observed in the results obtained from a quantitative analysis due to the type of reference substance used. The content of chalcones was varied and dependent on the species selected for analysis, namely from 22.01/21.08 mg/g in S. daphnoides clone 1095 (III) to 2.47/2.44 mg/g in S. daphnoides (II), collected from a natural habitat. Isosalipurposide (5) was determined in all the investigated species and clones of Salix, besides a number of naringenin derivatives. Separation of all flavonoids: flavanones - naringenin (1), naringenin (+)-5-O-glucoside (2), (-)-5-O-glucoside (3), 7-O-glucoside (4) and chalcones (5 and 6) was performed under gradient elution with the same solvents and changes in ACN concentration from 2% to 37% (tG 60 min). The total amount of flavanones ranged from 4.69 mg/g in S. purpurea clone 1132 (VII) to 41.93 mg/g in S. purpuea (VIII) from Herbapol Wrocław.

  3. Improved homopolymer separation to enable the application of 1H NMR and HPLC for the determination of the reaction parameters of the graft copolymerization of acrylic acid onto starch.

    PubMed

    Witono, Judy R; Marsman, Jan Henk; Noordergraaf, Inge-Willem; Heeres, Hero J; Janssen, Leon P B M

    2013-04-01

    Graft copolymers of starch with acrylic acid are a promising green, bio based material with many potential applications. The grafting of acrylic acid onto cassava starch in an aqueous medium initiated by Fenton's reagent has been studied. Common grafting result parameters are add-on (yield) and graft efficiency (selectivity). However, the analysis of the reaction products and an accurate determination of these parameters stand or fall with a complete separation of the entangled but ungrafted homopolymer from the grafted product. Therefore, this separation is the core of the newly developed analytical procedure. An appropriate solvent has been selected with dedicated testing from the range methanol, ethanol, acetone, dioxane, 2-propanol, and 1-propanol. Acetone showed the best performance in many respects. It has a high dissolving power for the homopolymer, as well as the highest yield of precipitation for the starch derivatives and it is the most economical in use. After the successful separation, the precipitated graft copolymers could be analyzed quantitatively by nuclear magnetic resonance. The liquid with homopolymer and unreacted monomer was analyzed by high pressure liquid chromatography. Proof of grafting has been found by FTIR and TGA analyses. The mass balance calculation shows a systematic error which appears fairly consistent: 18.0±2.5 wt%. This was used as a correction factor in the calculation of the grafting parameters but more importantly, it means that the method we developed has a high level of repeatability, in the order of 97%. PMID:23435285

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

    PubMed

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

    2011-02-20

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

  5. Assessment of the hemolysis and endothelial cell cytotoxicity induced by residual linear alkylbenzene sulfonates on pharmaceutical rubber stoppers based on HPLC-ESI-MS.

    PubMed

    Li, Xianghui; Qiu, Lu; Li, Yueyue; Chen, Xiaofei; Zhu, Zhenyu; Chai, Yifeng

    2015-09-01

    This study was designed to develop a high-performance liquid chromatographic-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method for quantitative determination of residual surfactant linear alkylbenzene sulfonate (LAS) compounds on pharmaceutical rubber stoppers. An HPLC-ESI-MS method was developed for separation and determination of five LAS homologs (C10-C14) under gradient conditions using methanol and ammonium acetate as mobile phases. Hemolysis activity of residual LAS compounds was analyzed by spectrophotometry. Expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in human umbilical vein endothelial cells (HUVECs) after LAS compound treatment was examined by enzyme-linked sorbent assay. LAS compounds were well separated and determined by the established gradient conditions. The linear range was 0.05-8 µg/mL with correlation coefficients ≥0.997. Recoveries were from 73 to 134% and the relative standard deviation was <13.7%. There was a correlation between hemolysis rate and LAS compounds concentration when it was ≥0.8 µg/cm(2). LAS compounds decreased the viability of HUVECs and promoted the production of IL-6 and TNF-α. The developed analytical method was successful for quantitative determination of residual LAS compounds on pharmaceutical rubber stoppers and it is important to monitor and control the amount of LAS compounds on rubber stoppers.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Development and Validation of a RP-HPLC Method for Determination of Related Substances and Degradants in Entacapone.

    PubMed

    Purnachand, Dasari; Veerareddy, Arava; Ramadevi, Bhoomireddy; Kameswarrao, Ch V S L; Madhusudhanreddy, Bethi

    2016-09-01

    A new reverse phase-liquid chromatography (RP-HPLC) method has been developed for simultaneous determination of entacapone and its pharmacopoeia impurities, in-house impurities and degradation impurities (total 17 analytes). Chromatographic separation was achieved on a C18 column (size: 250 × 4.6 mm; 5 µm particle size) at a flow rate of 1.0 mL/min with 210 nm detection. The mobile phase (MP) consists of 1.361 g of potassium di-hydrogen phosphate and 1.742 g of di-potassium phosphate in 1.0 L water, pH adjusted to 2.5 with ortho phosphoric acid (MP-A) and acetonitrile (MP-B) through gradient elution. The product was subjected to stress conditions such as acid, base, peroxide, thermal and photolytic degradation. Two new impurities above 2% level were observed and isolated through preparative HPLC and well characterized. However, no interference observed due to degradation impurities and entacapone and its EP impurities, in-house impurities. As part of the method validation, specificity, limit of detection, limit of quantitation (LOQ), linearity, accuracy, precision, robustness and ruggedness were determined. LOQ values were achieved between 0.01 and 0.04%. Good linearity (r(2) > 0.99) was obtained ranging from LOQ to 150%. Recovery was verified for all impurities at concentrations ranging from LOQ to 150%. Hence, a newly developed RP-HPLC method was capable for well separation of all analytes with acceptable resolution and tailing factor. PMID:27165569

  8. Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations.

    PubMed

    Dousa, Michal; Gibala, Petr

    2010-01-01

    A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.

  9. Determination of γ-Aminobutyric Acid (GABA) in Rambutan Fruit cv. Rongrian by HPLC-ELSD and Separation of GABA from Rambutan Fruit Using Dowex 50W-X8 Column.

    PubMed

    Meeploy, Maneerat; Deewatthanawong, Rujira

    2016-03-01

    A high-performance liquid chromatography method coupled with an evaporative light scattering detector (ELSD) was validated for the determination of γ-aminobutyric acid (GABA) in rambutan fruit without any sample pretreatment or derivatization. In the concentration range of 0.05-1.0 mg/mL GABA, the ELSD response was linear with a correlation coefficient (r) >0.999. Limit of detection and limit of quantitation were found to be 0.7 and 2.0 µg/mL, respectively. The method enabled the complete separation of GABA in the aqueous extract of rambutan flesh from the impurity peaks at 45.7 min. The recoveries of sample added GABA were obtained in the range of 92.0-99.3%. Intraday and interday relative standard deviations were <5.3%. Repeatability of the extraction process showed the acceptable precision. From the analysis of GABA content in rambutan flesh, 0.71 ± 0.23 mg of GABA was found in 1 g fresh weight. The recovery of GABA after passing through the Dowex 50W-X8 column was 96.65%. The analytical methodology could be potentially applied to the detection and quantification of GABA in other fruits and complex matrices when a sufficient quantity is available. PMID:26590236

  10. Simple isocratic method for simultaneous determination of caffeine and catechins in tea products by HPLC.

    PubMed

    Fernando, Chamira Dilanka; Soysa, Preethi

    2016-01-01

    Tea is a popular beverage almost all over the world. Many studies show that tea consumption is closely associated with positive health impact. Most of the HPLC methods used for the determination of tea constituents include gradient elution systems which involve expensive instrumentation. The objective of this study was to develop a simple, rapid precise and low cost HPLC method for the separation and quantification of catechins and caffeine in tea (Camellia sinensis). The method utilizes a phenyl column (2.1 × 150 mm) with a UV-detector (280 nm) where excellent chromatographic separation of tea components i.e. gallic acid (GA), caffeine (Caf), epicatechin (EC) and (-)-epigallocatechin gallate (EGCG) was achieved. The isocratic elution system of acetonitrile, glacial acetic acid and deionized water (8:1:91 v/v/v) at a flow rate of 0.5 mL/min was involved. This method produced excellent accuracy and precision. Within run and between run precision was less than 7.5 %. The equations for calibration curves were y = 0.117 (±0.010)x + 0.173 (±0.024), y = 0.100 (±0.003)x + 0.045 (±0.019), y = 0.016 (±0.001)x + 0.006 (±0.004), y = 0.025 (±0.001)x-0.025 (±0.007) for GA, Caf, EC and EGCG respectively. The method validation parameters prove that the method is efficient, a simple and adequate for the quantitative determination of principal components in tea samples. PMID:27429880

  11. Capillary electrophoresis as an orthogonal technique in HPLC method validation.

    PubMed

    Jimidar, M Ilias; De Smet, Maurits; Sneyers, Rudy; Van Ael, Willy; Janssens, Willy; Redlich, Dirk; Cockaerts, Paul

    2003-01-01

    High-performance liquid chromatography is usually used to assay the main compound and organic impurity content of drug substance and drug product during pharmaceutical development. A crucial validation parameter of these methods is specificity--the ability to unequivocally assess the analyte in the presence of component expected to be present. Typically, these include impurities, degradation products, and matrices. Besides adequate chromatographic separation with sufficient selectivity, additional 2- or 3-D spectroscopic or chromatographic tools are frequently necessary for this purpose. In our current practice, HPLC is used with ultraviolet photodiode array detection and on-line mass spectrometry (LC-UVDAD-MS) during the assessment of specificity. Although this approach is very powerful and can solve the majority of problems, separation of isomers of the main compound is still difficult. Since HPLC usually cannot offer the required selectivity and because of the similar molecular weights, structural isomers are not specifically detected using LC-MS. Capillary electrophoresis, on the other hand, offers high separation efficiency and can be applied as an adjunct to HPLC. Therefore, a set of highly selective CE methods is used orthogonally in the specificity assessment of HPLC methods.

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

    PubMed

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

    2014-02-26

    Chlorophylls and their green and olive-brown derivatives were successfully separated from culinary herb extracts by HPLC with photodiode-array and mass spectrometry detection. The method involved a ternary gradient elution and reverse-phase separation conditions capable of resolving 24 different pigments (2 chlorophylls and 22 of their derivatives) of different polarities within 28 min. The method was applied to monitor color changes in 50 samples of culinary aromatic herbs subjected to five different drying treatments. Of the 24 pigments, 14 were key to understanding the differences between the primary degradation pathways of chlorophyll a and chlorophyll b in culinary herbs during drying processes. A color degradation ladder based on the total molar percentage of all the remaining green pigments was also proposed as a tool to measure the impact of drying treatments on aromatic herb visual aspects. PMID:24483726

  13. Development and validation of a stability-indicating HPLC method for simultaneous determination of salicylic acid, betamethasone dipropionate and their related compounds in Diprosalic Lotion.

    PubMed

    Shou, Minshan; Galinada, Wilmer A; Wei, Yu-Chien; Tang, Qinglin; Markovich, Robert J; Rustum, Abu M

    2009-10-15

    Diprosalic Lotion is an anti-inflammatory drug product that contains salicylic acid and betamethasone dipropionate as active pharmaceutical ingredients (APIs). A reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for simultaneous determination of salicylic acid, betamethasone dipropionate, and their related compounds in Diprosalic Lotion. A 150 mm x 4.6 mm I.D. YMC J'sphere ODS-H80 column at 35 degrees C and UV detection at 240 nm was used. A gradient elution was employed using 0.05% (v/v) methanesulfonic acid solution and acetonitrile as mobile phases. A total of thirty three compounds from Diprosalic Lotion samples were separated in 38 min. The stability-indicating capability of this method has been demonstrated by the adequate separation of all the impurities and degradation products in expired stability samples of Diprosalic Lotion. The method was validated as per the current ICH guidelines.

  14. Effects of berberine and pomegranate seed oil on plasma phospholipid metabolites associated with risks of type 2 diabetes mellitus by U-HPLC/Q-TOF-MS.

    PubMed

    Wu, Xia; Li, Yan; Wang, Qiu; Li, Weimin; Feng, Yifan

    2015-12-15

    A rapid and reliable ultra-performance liquid chromatography coupled with electrospray ionization/quadrupole-time-of-flight mass spectrometry (U-HPLC/Q-TOF-MS) has been firstly used to analyze the changes of plasma phospholipids, in type 2 diabetes mellitus (T2DM) mice after administration of berberine and pomegranate seed oil (PSO). The separation of plasma phospholipids was carried out on an Acquity U-HPLC BEH C18 column (2.1mm×50mm, 1.7μm, Waters) by linear gradient elution using a mobile phase consisting of 10mM ammonium formate in water and acetonitrile: isopropanol (1:1, v/v) mixed solution added by 0.25% water and 10mM ammonium formate. The method demonstrated a good precision and reproducibility. Linear regression analysis showed a good linearity. And potential biomarkers were discovered based on their mass spectra and chemometrics methods. The results demonstrated that the proposed U-HPLC/Q-TOF-MS method was successfully applied to analyze the dynamic changes of phospholipids components in plasma of T2DM mice after drug treatment and could provide a useful data base for meriting further study in humans and investigating pharmacological actions of drugs.

  15. An HPLC-ELSD Method for the Determination of Triterpenes in Sorbus decora and Sorbus americana Bark Used by the Eeyou Istchee Cree First Nation.

    PubMed

    Saleem, Ammar; Liu, Rui; Guerrero-Analco, José A; Bailie, Anna; Foster, Brian; Cuerrier, Alain; Johns, Timothy; Haddad, Pierre S; Arnason, John Thor

    2016-09-01

    Sorbus decora and Sorbus americana are used traditionally as medicine by the Eeyou Istchee Cree First Nation of the James Bay region of Quebec, Canada. Because the ethanol extracts of the bark and the isolated terpenes of these plants have shown promising in vivo antidiabetic effects, an analytical method was developed and validated by RP-HPLC-ELSD for the identification and quantification of eight lupane- and ursane-type terpenes. The extraction method reproducibly recovered the compounds above 70 % and the chromatographic separation of betulin, 23-hydroxy-betulin, 23,28-dihydroxylupan-20(29)-ene-3β-caffeate, betulinic acid, α-amyrin, uvaol, 3β,23,28-trihydroxy-12-ursene, and 23,28-dihydroxyursan-12-ene-3β-caffeate was achieved within 27 min by linear gradient. The method produced highly reproducible quantitative data at interday and intraday levels. The limits of detection were in the ng level on-column with remarkable range and linearity. The target compounds were present at mg levels in the populations, collected from inland (Mistissini and Nemaska) and costal (Waskagnish and Chisasibi) Cree communities of northern Quebec. A triterpene, 23-hydroxybetulin, was the most abundant, while betulinic acid and uvaol were minor constituents. Overall, HPLC-ELSD analyses produced very similar profiles and contents of the eight compounds in the plants collected from four geographic locations. The developed HPLC-ELSD method can be used as a targeted analysis of triterpenes in these medicinal plants. PMID:27163231

  16. Effects of berberine and pomegranate seed oil on plasma phospholipid metabolites associated with risks of type 2 diabetes mellitus by U-HPLC/Q-TOF-MS.

    PubMed

    Wu, Xia; Li, Yan; Wang, Qiu; Li, Weimin; Feng, Yifan

    2015-12-15

    A rapid and reliable ultra-performance liquid chromatography coupled with electrospray ionization/quadrupole-time-of-flight mass spectrometry (U-HPLC/Q-TOF-MS) has been firstly used to analyze the changes of plasma phospholipids, in type 2 diabetes mellitus (T2DM) mice after administration of berberine and pomegranate seed oil (PSO). The separation of plasma phospholipids was carried out on an Acquity U-HPLC BEH C18 column (2.1mm×50mm, 1.7μm, Waters) by linear gradient elution using a mobile phase consisting of 10mM ammonium formate in water and acetonitrile: isopropanol (1:1, v/v) mixed solution added by 0.25% water and 10mM ammonium formate. The method demonstrated a good precision and reproducibility. Linear regression analysis showed a good linearity. And potential biomarkers were discovered based on their mass spectra and chemometrics methods. The results demonstrated that the proposed U-HPLC/Q-TOF-MS method was successfully applied to analyze the dynamic changes of phospholipids components in plasma of T2DM mice after drug treatment and could provide a useful data base for meriting further study in humans and investigating pharmacological actions of drugs. PMID:26590882

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

    PubMed

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

    2010-07-26

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

  18. Centrifugal partition chromatography elution gradient for isolation of sesquiterpene lactones and flavonoids from Anvillea radiata.

    PubMed

    Destandau, Emilie; Boukhris, Meryem Alaoui; Zubrzycki, Sandrine; Akssira, Mohamed; Rhaffari, Lhoucine El; Elfakir, Claire

    2015-03-15

    An innovative procedure coupling pressurized solvent extraction and centrifugal partition chromatography (CPC) used in linear gradient elution mode was developed to isolate two pure germacranolides (9α-hydroxyparthenolide and 9β-hydroxyparthenolide) and to separate flavonoids (nepetin, isorhamnetin and jaceosidin) and chlorophyll pigments from aerial parts of Anvillea radiata (Coss.&Durieu). The two main germacranolides recovered using this method represent 2 and 5% of the dried plant material respectively. These molecules were extracted using accelerated solvent extraction with chloroform. After optimization of the CPC method, a two-phase solvent system composed of heptane/ethyl acetate/methanol/water (1:5:1:5 v/v/v/v) was employed in descending mode to isolate the germacranolides. Then the lower phase of a heptane/ethyl acetate/methanol/water (6:5:6:5 v/v/v/v) system was pumped in descending mode to generate a linear elution gradient, progressively decreasing the mobile phase polarity, that enabled the flavonoid compounds to be separated in the same run. The efficiency of the preparative separation was controlled through RP-HPLC analysis of the obtained fractions using UV, evaporative light scattering and mass spectrometry detection. The structural identification of the two germacranolides purified over 99% was established by (1)H NMR and (13)C NMR. The least abundant flavonoids were identified by mass spectrometry. PMID:25647341

  19. Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry.

    PubMed

    McCoy, Leslie F; Scholl, Peter F; Sutcliffe, Anne E; Kieszak, Stephanie M; Powers, Carissa D; Rogers, Helen S; Gong, Yun Yun; Groopman, John D; Wild, Christopher P; Schleicher, Rosemary L

    2008-07-01

    Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B(1). In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

  20. Separation of bimodal high density polyethylene using multidimensional high temperature liquid chromatography.

    PubMed

    Prabhu, K N; Brüll, R; Macko, T; Remerie, K; Tacx, J; Garg, P; Ginzburg, A

    2015-11-01

    High-temperature two-dimensional liquid chromatography (HT 2D-LC) using HT-HPLC as first dimension and HT-SEC as second dimension holds enormous potential to investigate the distribution according to molar mass and chemical composition of bimodal high density polyethylene (BiHDPE), as it avoids drawbacks of crystallization-based techniques. In this study, we have stepwise optimized the chromatographic parameters of 1D, comprising gradient slope and temperature, using model homo- and copolymers of ethylene with the aim to minimize the impact of molar mass on the compositional separation. Then the HT-HPLC was hyphenated to HT-SEC and optimum conditions for the volume of the sample transfer loop were probed with regard to the resolution of BiHDPE into the individual constituents HDPE and LLDPE. A particular important aspect was the use of infrared (IR) detection, and the demands it puts on the chromatographic aspects: We have shown that IR detection can be successfully applied in HT 2D-LC of BiHDPE, which is broadly distributed with regard to short chain branching and molar mass, only when the separation in 2D is optimized with regard to chromatographic resolution. As final result a bimodality is evident in the contour and the 3D surface plots as well as in both HPLC and SEC projections generated from HT 2D-LC. PMID:26435312

  1. Separation of bimodal high density polyethylene using multidimensional high temperature liquid chromatography.

    PubMed

    Prabhu, K N; Brüll, R; Macko, T; Remerie, K; Tacx, J; Garg, P; Ginzburg, A

    2015-11-01

    High-temperature two-dimensional liquid chromatography (HT 2D-LC) using HT-HPLC as first dimension and HT-SEC as second dimension holds enormous potential to investigate the distribution according to molar mass and chemical composition of bimodal high density polyethylene (BiHDPE), as it avoids drawbacks of crystallization-based techniques. In this study, we have stepwise optimized the chromatographic parameters of 1D, comprising gradient slope and temperature, using model homo- and copolymers of ethylene with the aim to minimize the impact of molar mass on the compositional separation. Then the HT-HPLC was hyphenated to HT-SEC and optimum conditions for the volume of the sample transfer loop were probed with regard to the resolution of BiHDPE into the individual constituents HDPE and LLDPE. A particular important aspect was the use of infrared (IR) detection, and the demands it puts on the chromatographic aspects: We have shown that IR detection can be successfully applied in HT 2D-LC of BiHDPE, which is broadly distributed with regard to short chain branching and molar mass, only when the separation in 2D is optimized with regard to chromatographic resolution. As final result a bimodality is evident in the contour and the 3D surface plots as well as in both HPLC and SEC projections generated from HT 2D-LC.

  2. A Straightforward and Highly Efficient Precipitation/On-pellet Digestion Procedure Coupled to a Long Gradient Nano-LC Separation and Orbitrap Mass Spectrometry for Label-free Expression Profiling of the Swine Heart Mitochondrial Proteome

    PubMed Central

    Duan, Xiaotao; Young, Rebecca; Straubinger, Robert M.; Page, Brian J.; Cao, Jin; Wang, Hao; Yu, Haoying; Canty, John M.; Qu, Jun

    2009-01-01

    For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the

  3. On gradient field theories: gradient magnetostatics and gradient elasticity

    NASA Astrophysics Data System (ADS)

    Lazar, Markus

    2014-09-01

    In this work, the fundamentals of gradient field theories are presented and reviewed. In particular, the theories of gradient magnetostatics and gradient elasticity are investigated and compared. For gradient magnetostatics, non-singular expressions for the magnetic vector gauge potential, the Biot-Savart law, the Lorentz force and the mutual interaction energy of two electric current loops are derived and discussed. For gradient elasticity, non-singular forms of all dislocation key formulas (Burgers equation, Mura equation, Peach-Koehler stress equation, Peach-Koehler force equation, and mutual interaction energy of two dislocation loops) are presented. In addition, similarities between an electric current loop and a dislocation loop are pointed out. The obtained fields for both gradient theories are non-singular due to a straightforward and self-consistent regularization.

  4. HPLC determination of imidazole antimycotis in antidandruff cosmetic products.

    PubMed

    Gagliardi, Luigi; De Orsi, Daniela; Chimenti, Paola; Porra', Rita; Tonelli, Domenica

    2003-08-01

    A simple HPLC method for the determination of imidazole antimycotics in cosmetic antidandruff formulations has been developed. HPLC was carried out on a Discovery RP-Amide C16 column and spectrophotometric detection was performed at 220 nm. The initial mobile phase was a mixture of acetonitrile and aqueous 10(-3) M NaClO4 (pH 3.0) in the ratio of 15:85 (v/v); then a linear gradient up to 46% acetonitrile in 70 min, and up to 50% in 80 min. The extraction procedure has been validated by analyzing samples of shampoo and lotion spiked with 1% of the active principles. The recoveries were greater than 95% and the reproducibility was within 3%.

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

    PubMed Central

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

    2016-01-01

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

  6. HPLC METHOD DEVELOPMENT, VALIDATION AND IMPURITY CHARACTERIZATION FOR AN ANTITUMOR HSP90 INHIBITOR -- PU-H71 (NSC 750424)

    PubMed Central

    Liu, Mingtao; Wang, Jennie; Wu, Xiaogang; Wang, Euphemia; Baptista, Diego; Scott, Brendan; Liu, Paul

    2013-01-01

    An HPLC method for the assay of the heat shock protein 90 inhibitor, PU-H71 (NSC 750424), has been developed and validated. The stress testing of PU-H71 was carried out in accordance with ICH guidelines Q1A (R2) under aqueous, acidic, alkaline, oxidative, thermolytic and photolytic conditions. The separation of PU-H71 from its impurities and degradation products was achieved within 50 min on a Mac-Mod ACE 3 C18 column (150 mm × 4.6 mm i.d., 3 μm) with a gradient mobile phase comprising 20–95% acetonitrile in water, with 0.1% trifluroacetic acid in both phases. LC - Quadruple TOF/MS was used to obtain accurate mass data on various components as well as on their fragments for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.1–0.3 mg/mL, r ≥ 0.9998), accuracy (recovery 99.7–101.1%), precision (intra-lab RSD ≤ 1.39%, inter-lab RSD ≤ 0.91%), sensitivity (LOD 0.08 μg/mL), and ruggedness. The developed method was suitable for the assay and stability monitoring of PU-H71 drug substance. PMID:24252723

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

    PubMed

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

    2015-01-01

    Tuberculosis treatment consists of a fixed dose combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PYZ), and ethambutol hydrochloride (EMB). The combined treatment using various drugs is necessary for patient curing, without recrudescence, and for prevention of drug-resistant mutants, which may occur during treatment. An HPLC-diode array detector (DAD) method for the simultaneous determination of RIF, INH, PYZ, and EMB in fixed dose combination tablets was developed and validated. Chromatographic experiments were performed on an Agilent 1200 HPLC system, and the separation was carried out on a Purospher STAR RP18e (250×4.6 mm id, 5 μm, Merck) analytical column. Gradient elution was carried out with a mobile phase of 20 mM monobasic sodium phosphate buffer with 0.2% triethylamine (pH 7.0) and acetonitrile at a flow rate of 1.5 mL/min. The total run time was 12 min, and the re-equilibration time was 5 min. EMB detection was performed at 210 nm, and RIF, INH, and PYZ were detected at 238 nm, using a DAD. The method proved to be specific, linear (r2>0.99), precise (RSD<2%), accurate, and robust and may be applied to the QC analysis of pharmaceutical formulations. PMID:26525241

  8. HPLC method development, validation and impurity characterization for an antitumor Hsp90 inhibitor-PU-H71 (NSC 750424).

    PubMed

    Liu, Mingtao; Wang, Jennie; Wu, Xiaogang; Wang, Euphemia; Baptista, Diego; Scott, Brendan; Liu, Paul

    2014-02-01

    An HPLC method for the assay of the heat shock protein 90 inhibitor, PU-H71 (NSC 750424), has been developed and validated. The stress testing of PU-H71 was carried out in accordance with ICH guidelines Q1A (R2) under aqueous, acidic, alkaline, oxidative, thermolytic and photolytic conditions. The separation of PU-H71 from its impurities and degradation products was achieved within 50min on a Mac-Mod ACE 3 C18 column (150mm×4.6mm i.d., 3μm) with a gradient mobile phase comprising 20-95% acetonitrile in water, with 0.1% trifluroacetic acid in both phases. LC-quadrupole TOF/MS was used to obtain accurate mass data on various components as well as on their fragments for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.1-0.3mg/mL, r≥0.9998), accuracy (recovery 99.7-101.1%), precision (intra-lab RSD≤1.39%, inter-lab RSD≤0.91%), sensitivity (LOD 0.08μg/mL), and ruggedness. The developed method was suitable for the assay and stability monitoring of PU-H71 drug substance. PMID:24252723

  9. Development and validation of a confirmatory HPLC method for simultaneous determination of Sudan dyes in animal tissues and eggs.

    PubMed

    Wu, Yin-Liang; Li, Cun; Xia, Xi; Liu, Yong-Jun; Shen, Jian-Zhong

    2010-01-01

    A simple and sensitive high-performance liquid chromatography (HPLC) analytical method for the simultaneous determination of six Sudan dyes (Sudan Red G, Sudan I, Sudan II, Sudan III, Sudan Red 7B, Sudan IV) in animal tissues and eggs was developed. Samples were extracted with acetonitrile followed by a cleanup using a C(18) solid-phase extraction column. Chromatographic separation was achieved on a Zorbax SB-C(18) column under gradient conditions. The analytes were detected at 510 nm by HPLC with diode array detection. Specificity, decision limit (CCalpha), detection capacity (CCbeta), accuracy, and precision were determined during validation process. Recoveries for six Sudan dyes from three animal tissues and eggs were 77.2-98.0% with excellent relative standard deviations. CCalpha and CCbeta were in the range of 7.7-9.0 microg/kg and 9.1-10.3 microg/kg, respectively. The limits of quantitations were between 12.8 microg/kg and 15.0 microg/kg.

  10. [Quantitative determination of 5 active ingredients in different harvest periods of Ligusticum chuanxiong by HPLC].

    PubMed

    Liu, Jin-Liang; Fan, Qiao-Jia; Zheng, Shun-Lin; Tan, Jie; Zhou, Juan; Yuan, Ji-Chao; Yang, Shi-Min; Kong, Fan-Lei

    2014-05-01

    A simple and quick method is described for the determination of ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A and ligustilide in rhizomes of Ligusticum chuanxiong. The 5 active ingredients in the sample was extracted using 40% ethanol and analyzed by reversed-phase high performance liquid chromatography (HPLC). Chromatography separation was performed using Agilent 1100 series HPLC system with a Symmetry C18 column and gradient elution with a mixture of three solvents : solvent A, acetonitrile, solvent B, methanol and solvent C, 1% aqueous acetic acid, 0 min to 5 min A: B: C 20: 40: 40, 5 min to 30 min A: B: C 60 to 100 : 0 : 40 to 0. The effluent was monitored using a VWD detector set at 321 nm (0-4.3 min) and 275 nm (4.31-30 min). The flow rate was set at 1 mL x min(-1) and the injection volume was 10 microL. The column temperature was maintained at 35 degrees C. The calibration curve was linear (r > or = 0.99) over the tested ranges. The average recovery was 94.44%-103.1% (n = 6). The method has been successfully applied to the analysis in different harvest periods of L. chuanxiong samples. In this paper, single-factor randomized block design to study the 5 components content of L. chuanxiong on ten collecting stages. For the L. chuanxiong collected from April 15th to May 30rd, the content of 5 ingredients increased primarily, and then decreased. Determine the appropriate harvest time has important significance to the promotion of the quality of L. chuanxiong. PMID:25095378

  11. Preliminary Study of High Resolution HPLC Analytical Method for Sedimentary Pigments Based on Coupled C8 Columns

    NASA Astrophysics Data System (ADS)

    Yao, P.; Yu, Z.; Deng, C.; Liu, S.; Zhao, J.

    2008-05-01

    The pigments in marine water columns can provide accurate estimates of community composition and abundance of phytoplankton. In addition, the sedimentary pigments, especially the derivatives of chlorophyll such as pyrophaeophytins, pyrophaeophorbides and steryl chlorin esters (SCEs) formed during early diagenesis can also provide information on the primary producer community and the changes in paleoproductivity. Accordingly, analysis of pigments and their derivatives is of great importance for oceanography, limnology and geochemistry. Many methods have been developed for the separation of chlorophylls, carotenoids and their derivatives derived from phytoplankton and water column samples using high-performance liquid chromatography (HPLC). Methods widely cited in the literatures include those developed by Wright et al. (1991) and Zapata et al. (2000). Both methods use reversed-phase columns, but C18 column was employed in Wright et al. (1991) and C8 column in Zapata et al. (2000). However, evident coelutions are observed in published works. This will particularly cause problematic identification and quantification in dealing with sedimentary pigments which are highly complex and often display a broad range in polarity. Clearly, it is necessary to improve the separation of the complex pigments if the information carried by the pigments is to be used fully. Coupled C18 columns were used in the HPLC method developed by Airs et al. (2001) for the analysis of complex pigment distributions. Improved chromatographic resolution, more pigment components and novel bacteriochlorophyll derivatives were obtained by this method. It indicates a new road for HPLC method development. C8 column has shorter carbon chains than that of C18 column and can provide less retention of apolar compounds which is of particular advantaged to hydrophobic chlorophyll a, b and their derivatives. That is one of the reasons why the C8 method developed by Zapata et al. (2000) is admittedly better than

  12. Simultaneous determination of eight biologically active thiol compounds using gradient elution-liquid chromatography with Coul-Array detection.

    PubMed

    Petrlova, Jitka; Mikelova, Radka; Stejskal, Karel; Kleckerova, Andrea; Zitka, Ondrej; Petrek, Jiri; Havel, Ladislav; Zehnalek, Josef; Vojtech, Adam; Trnkova, Libuse; Kizek, Rene

    2006-05-01

    The most active form of sulfur in biomolecules is the thiol group, present in a number of biologically active compounds. Here we present a comprehensive study of thiol analysis using flow injection analysis/HPLC with electrochemical detection. The effect of different potentials of working electrodes, of organic solvent contents in the mobile phase, and of isocratic and gradient elution on simultaneous determination of thiol compounds (cysteine, cystine, N-acetylcysteine, homocysteine, reduced and oxidised glutathione, desglycinephytochelatin, and phytochelatins) are described and discussed. These thiol compounds were well separated and detected under optimised HPLC-electrochemical detection conditions (mobile phase: 80 mM trifluoroacetic acid and methanol with a gradient profile starting at 97:3 (TFA:methanol), kept constant for the first 8 min, then decreasing to 85:15 during one minute, kept constant for 8 min, and finally increasing linearly up to 97:3 from 17 to 18 min; the flow rate was 0.8 mL/min, column and detector temperature 25 degrees C, and the electrode potential 900 mV). We were able to determine tens of femtomoles (3 S/N) of the thiols per injection (5 microL), except for phytochelatin5 whose detection limit was 2.1 pmole. This technique was consequently used for simultaneous determination of compounds of interest in biological samples (maize tissue and human blood serum). PMID:16830732

  13. Enhancing Centrifugal Separation With Electrophoresis

    NASA Technical Reports Server (NTRS)

    Herrmann, F. T.

    1986-01-01

    Separation of biological cells by coil-planet centrifuge enhanced by electrophoresis. By itself, coil-planet centrifuge offers relatively gentle method of separating cells under low centrifugal force in physiological medium that keeps cells alive. With addition of voltage gradient to separation column of centrifuge, separation still gentle but faster and more complete. Since separation apparatus contains no rotary seal, probability of leakage, contamination, corrosion, and short circuits reduced.

  14. Electrochemically Pretreated Carbon Microfiber Electrodes as Sensitive HPLC-EC Detectors

    PubMed Central

    Bartosova, Zdenka; Riman, Daniel; Jakubec, Petr; Halouzka, Vladimir; Hrbac, Jan; Jirovsky, David

    2012-01-01

    The paper focuses on the analysis and detection of electroactive compounds using high-performance liquid chromatography (HPLC) combined with electrochemical detection (EC). The fabrication and utilization of electrochemically treated carbon fiber microelectrodes (CFMs) as highly sensitive amperometric detectors in HPLC are described. The applied pretreatment procedure is beneficial for analytical characteristics of the sensor as demonstrated by analysis of the model set of phenolic acids. The combination of CFM with separation power of HPLC technique allows for improved detection limits due to unique electrochemical properties of carbon fibers. The CFM proved to be a promising tool for amperometric detection in liquid chromatography. PMID:22654586

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

    PubMed

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

    2011-01-01

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

  16. Methods and applications of HPLC-AMS (WBio 5)

    SciTech Connect

    Bucholz, B A; Clifford, A J; Duecker, S R; Lin, Y; Vogel, J S

    1999-09-29

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

  17. Isolation of Early and Late Endosomes by Density Gradient Centrifugation.

    PubMed

    de Araújo, Mariana E G; Lamberti, Giorgia; Huber, Lukas A

    2015-11-01

    Density gradient centrifugation is a common method for separating intracellular organelles. During centrifugation, organelles float or sediment until they reach their isopycnic position within the gradient. The density of an organelle depends on its content, size, shape, and the lipid:protein ratio. The degree of separation between different organelles will therefore be highly dependent on how different their isopycnic points are in a given buffer. Separation will also depend on the medium used to prepare the gradient, whether it is sucrose (the most common) or an alternative. Here we describe the use of both continuous and discontinuous (step) gradients to isolate endocytic organelles. PMID:26527762

  18. Prediction of peptide retention at different HPLC conditions from multiple linear regression models.

    PubMed

    Baczek, Tomasz; Wiczling, Paweł; Marszałł, Michał; Heyden, Yvan Vander; Kaliszan, Roman

    2005-01-01

    To quantitatively characterize the structure of a peptide and to predict its gradient retention time at given HPLC conditions three structural descriptors are used: (i) logarithm of the sum of retention times of the amino acids composing the peptide, log SumAA, (ii) logarithm of the van der Waals volume of the peptide, log VDW(Vol), (iii) and the logarithm of the peptide's calculated n-octanol-water partition coefficient, clog P. The log SumAA descriptor is obtained from empirical data for 20 natural amino acids, determined in a given HPLC system. The two other descriptors are calculated from the peptides' structural formulas using molecular modeling methods. The quantitative structure-retention relationships (QSRR), build by multiple linear regression, describe HPLC retention of peptide on a given chromatographic system on which the retention of the 20 amino acids was predetermined. A structurally diversified series of 98 peptides was employed. The predicted gradient retention times on several chromatographic systems were in good agreement with the experimental data. The QSRR equations, derived for a given system operated at variable gradient times and temperatures allowed for the prediction of peptide retention in that system. Matching the experimental HPLC retention to the theoretically predicted for a presumed peptide could facilitate original protein identification in proteomics. In conjunction with MS data, prediction of the retention time for a given peptide might be used to improve the confidence of peptide identifications and to increase the number of correctly identified peptides.

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

    PubMed

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

    2007-01-01

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

  20. Separation of maleic anhydride grafted polypropylene using multidimensional high-temperature liquid chromatography.

    PubMed

    Prabhu, K N; Macko, T; Brüll, R; Remerie, K; Tacx, J; Garg, P; Ginzburg, A

    2016-04-01

    Functionalization addresses a property gap of polyolefins and opens new perspectives due to improved surface properties in applications like composites (e.g., glass fiber reinforced polypropylene) and anti-corrosive coatings for metals. Various techniques have been developed to characterize functionalized polyolefins, yet no analytical approach addressing their chemical heterogeneity exists. Using High Temperature Size Exclusion Chromatography (HT-SEC) coupled to infrared spectroscopy we could show for two model samples of polypropylene grafted maleic anhydride (PP-g-MA), differing in their nominal MA content, that the grafting density increases with decreasing molar mass. Crystallization Analysis Fractionation (CRYSTAF) does not enable to separate these samples according to their composition to the extent required. Yet, when using High Temperature High Performance Liquid Chromatography (HT-HPLC), with either silica gel or Mica as stationary phase and a gradient mobile phase, a deformulation into a grafted and a non-grafted fraction could be achieved. This was confirmed by analyzing the eluted fractions by infrared spectroscopy. Hyphenating the separation according to composition with a separation according to molar mass (HT-HPLC x HT-SEC) enabled for the first time to reveal the bivariate distribution of PP-g-MA with regard to the molar mass and composition. Using on-line infrared detection quantitative information on the compositional and molar mass parameters of the individual fractions could be obtained. PMID:26961914

  1. Separation of maleic anhydride grafted polypropylene using multidimensional high-temperature liquid chromatography.

    PubMed

    Prabhu, K N; Macko, T; Brüll, R; Remerie, K; Tacx, J; Garg, P; Ginzburg, A

    2016-04-01

    Functionalization addresses a property gap of polyolefins and opens new perspectives due to improved surface properties in applications like composites (e.g., glass fiber reinforced polypropylene) and anti-corrosive coatings for metals. Various techniques have been developed to characterize functionalized polyolefins, yet no analytical approach addressing their chemical heterogeneity exists. Using High Temperature Size Exclusion Chromatography (HT-SEC) coupled to infrared spectroscopy we could show for two model samples of polypropylene grafted maleic anhydride (PP-g-MA), differing in their nominal MA content, that the grafting density increases with decreasing molar mass. Crystallization Analysis Fractionation (CRYSTAF) does not enable to separate these samples according to their composition to the extent required. Yet, when using High Temperature High Performance Liquid Chromatography (HT-HPLC), with either silica gel or Mica as stationary phase and a gradient mobile phase, a deformulation into a grafted and a non-grafted fraction could be achieved. This was confirmed by analyzing the eluted fractions by infrared spectroscopy. Hyphenating the separation according to composition with a separation according to molar mass (HT-HPLC x HT-SEC) enabled for the first time to reveal the bivariate distribution of PP-g-MA with regard to the molar mass and composition. Using on-line infrared detection quantitative information on the compositional and molar mass parameters of the individual fractions could be obtained.

  2. [Multiresidue determination of quinolones in animal and fishery products by HPLC].

    PubMed

    Chonan, Takao; Fujimoto, Toru; Inoue, Maki; Tazawa, Teijiro; Ogawa, Hiroshi

    2008-06-01

    A simple and rapid multiresidue method was developed for the determination of twelve quinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid and sarafloxacin) in muscle, liver, chicken eggs, milk, prawn and rainbow trout. The quinolones were extracted from a sample with acetonitrile-water (95 : 5). A fifth part of the filtered extract was diluted with water to keep the acetonitrile ratio at ca. 60%, and passed through a C18 mini-column. The eluate was evaporated to dryness, and the residues were dissolved in methanol-water (30 : 70) for HPLC analysis. The quinolones were separated on a Inertsil ODS-3V column (4.6 mm i.d.x250 mm) with a gradient system of 0.1% phosphoric acid-acetonitrile as the mobile phase, with fluorescence detection.No interfering peak was found on the chromatograms of animal and fishery products, except for milk. The recoveries of the quinolones were over 60% from the animal and fishery products fortified at 0.1 microg/g, and the quantification limits of the quinolones were 0.005 microg/g. This proposed method was found to be effective and suitable for the screening of the quinolones in animal and fishery products.

  3. Absorbance detector based on a deep UV light emitting diode for narrow-column HPLC.

    PubMed

    Bui, Duy Anh; Bomastyk, Benjamin; Hauser, Peter C

    2013-10-01

    A detector for miniaturized HPLC based on deep UV emitting diodes and UV photodiodes was constructed. The measurement is accomplished by the transverse passage of the radiation from the light-emitting diode (LED) through fused-silica tubing with an internal diameter of 250 μm. The optical cell allows flexible alignment of the LED, tubing, and photodiode for optimization of the light throughput and has an aperture to block stray light. A beam splitter was employed to direct part of the emitted light to a reference photodiode and the Lambert-Beer law was emulated with a log-ratio amplifier circuitry. The detector was tested with two LEDs with emission bands at 280 and 255 nm and showed noise levels as low as 0.25 and 0.22 mAU, respectively. The photometric device was employed successfully in separations using a column of 1 mm inner diameter in isocratic as well as gradient elution. Good linearities over three orders of magnitude in concentration were achieved, and the precision of the measurements was better than 1% in all cases. Detection down to the low micromolar range was possible. PMID:23893947

  4. Simultaneous HPLC determination of 22 components of essential oils; method robustness with experimental design.

    PubMed

    Porel, A; Sanyal, Y; Kundu, A

    2014-01-01

    The aim of the present study was the development and validation of a simple, precise and specific reversed phase HPLC method for the simultaneous determination of 22 components present in different essential oils namely cinnamon bark oil, caraway oil and cardamom fruit oil. The chromatographic separation of all the components was achieved on Wakosil-II C18 column with mixture of 30 mM ammonium acetate buffer (pH 4.7), methanol and acetonitrile in different ratio as mobile phase in a ternary linear gradient mode. The calibration graphs plotted with five different concentrations of each component were linear with a regression coefficient R(2) >0.999. The limit of detection and limit of quantitation were estimated for all the components. Effect on analytical responses by small and deliberate variation of critical factors was examined by robustness testing with Design of Experiment employing Central Composite Design and established that this method was robust. The method was then validated for linearity, precision, accuracy, specificity and demonstrated to be applicable to the determination of the ingredients in commercial sample of essential oil.

  5. Simultaneous determination of 15 nitroimidazoles in cosmetics by HPLC coupled with electrospray ionization- tandem mass spectrometry.

    PubMed

    Meng, Xian-Shuang; Bai, Hua; Zhang, Qing; Lv, Qing; Chen, Yun-Xia; Ma, Hui-Juan; Li, Jing-Rui; Ma, Qiang

    2014-01-01

    A sensitive and reliable analytical method based on HPLC/MSIMS has been developed for the simultaneous determination of 15 nitroimidazoles in cosmetics. A diversity of cosmetic samples, including powder, lotion, shampoo, and cream were collected. The samples were ultrasonically extracted with aqueous methanol, and the extracts were then subjected to cleanup bySPE using an Oasis HLB cartridge followed by filtration with a 0.20 pm membrane filter. Afterwards, chromatographic separation was performed on an XSelect CSH C18 column (2.1 x 150 mm, 3.5 pm) maintained at 30°C within 15 min by a gradient of acetonitrile-0.1% aqueous formic acid solution at a flow rate of 0.25 mL/min. The mass spectrometric detection was carried, out using electrospray positive ionization under the multiple reaction monitoring mode. A good linearity was observed over the concentration range from 0.5 to 500 ng/mL. The intraday and interday precisions, which were investigated by determining all target compounds in cosmetics seven times/day and on 7 consecutive days, were below 5.00%. The mean recoveries at three spiked levels ranged from 80.42 to 100.83% with the RSDs from 0.45 to 9.02%. The LOQs were determined to be between 0.01 and 0.1 mg/kg. The method was sufficiently rapid, reliable, and sensitive for the determination of 15 nitroimidazoles in cosmetics. PMID:25632431

  6. Simultaneous Quantification of 13 Compounds in Guanxin Shutong Capsule by HPLC Method.

    PubMed

    Zhou, Feifei; Zhang, Lunhui; Gu, Liqiang; Zhang, Yuanyuan; Hou, Chenzhi; Bi, Kaishun; Chen, Xiaohui; Zhang, Huifen

    2016-07-01

    Guanxin Shutong (GXST) capsule, which is frequently used in clinical therapy, has a certain and positive therapeutic effect against coronary heart disease. However, the existing quality standard of GXST capsule is inadequate to control the quality of GXST capsule. In this paper, a new high-performance liquid chromatographic (HPLC) method for simultaneous determination of 13 compounds (gallic acid, danshensu, protocatechuic acid, procatechuic aldehyde, ellagic acid, rosmarinic acid, salvianolic acid A and salvianolic acid B, eugenol, dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA) in GXST capsule was developed and validated. The analytes were successfully separated and quantified with an Agilent TC-C18 column (250 × 4.6 mm, 5 µm) by gradient elution using 0.05% phosphoric acid and acetonitrile as mobile phase. The flow rate was 1 mL/min and the detection wavelength was set at 280 nm. All the compounds showed good linearity (r > 0.9991) in a relatively wide concentration range. The intra-and the inter-day variability were in the range of 0.85-2.68 and 1.47-2.86%, respectively. The recoveries of the selected compounds were in the range of 95.24-104.75%. This method was successfully applied to quantify the 13 components in GXST capsule and was conducive to controlling the quality of GXST capsule. PMID:26969683

  7. Simultaneous determination of 15 nitroimidazoles in cosmetics by HPLC coupled with electrospray ionization- tandem mass spectrometry.

    PubMed

    Meng, Xian-Shuang; Bai, Hua; Zhang, Qing; Lv, Qing; Chen, Yun-Xia; Ma, Hui-Juan; Li, Jing-Rui; Ma, Qiang

    2014-01-01

    A sensitive and reliable analytical method based on HPLC/MSIMS has been developed for the simultaneous determination of 15 nitroimidazoles in cosmetics. A diversity of cosmetic samples, including powder, lotion, shampoo, and cream were collected. The samples were ultrasonically extracted with aqueous methanol, and the extracts were then subjected to cleanup bySPE using an Oasis HLB cartridge followed by filtration with a 0.20 pm membrane filter. Afterwards, chromatographic separation was performed on an XSelect CSH C18 column (2.1 x 150 mm, 3.5 pm) maintained at 30°C within 15 min by a gradient of acetonitrile-0.1% aqueous formic acid solution at a flow rate of 0.25 mL/min. The mass spectrometric detection was carried, out using electrospray positive ionization under the multiple reaction monitoring mode. A good linearity was observed over the concentration range from 0.5 to 500 ng/mL. The intraday and interday precisions, which were investigated by determining all target compounds in cosmetics seven times/day and on 7 consecutive days, were below 5.00%. The mean recoveries at three spiked levels ranged from 80.42 to 100.83% with the RSDs from 0.45 to 9.02%. The LOQs were determined to be between 0.01 and 0.1 mg/kg. The method was sufficiently rapid, reliable, and sensitive for the determination of 15 nitroimidazoles in cosmetics.

  8. Absorbance detector based on a deep UV light emitting diode for narrow-column HPLC.

    PubMed

    Bui, Duy Anh; Bomastyk, Benjamin; Hauser, Peter C

    2013-10-01

    A detector for miniaturized HPLC based on deep UV emitting diodes and UV photodiodes was constructed. The measurement is accomplished by the transverse passage of the radiation from the light-emitting diode (LED) through fused-silica tubing with an internal diameter of 250 μm. The optical cell allows flexible alignment of the LED, tubing, and photodiode for optimization of the light throughput and has an aperture to block stray light. A beam splitter was employed to direct part of the emitted light to a reference photodiode and the Lambert-Beer law was emulated with a log-ratio amplifier circuitry. The detector was tested with two LEDs with emission bands at 280 and 255 nm and showed noise levels as low as 0.25 and 0.22 mAU, respectively. The photometric device was employed successfully in separations using a column of 1 mm inner diameter in isocratic as well as gradient elution. Good linearities over three orders of magnitude in concentration were achieved, and the precision of the measurements was better than 1% in all cases. Detection down to the low micromolar range was possible.

  9. Simultaneous HPLC Determination of 22 Components of Essential Oils; Method Robustness with Experimental Design

    PubMed Central

    Porel, A.; Sanyal, Y.; Kundu, A.

    2014-01-01

    The aim of the present study was the development and validation of a simple, precise and specific reversed phase HPLC method for the simultaneous determination of 22 components present in different essential oils namely cinnamon bark oil, caraway oil and cardamom fruit oil. The chromatographic separation of all the components was achieved on Wakosil–II C18 column with mixture of 30 mM ammonium acetate buffer (pH 4.7), methanol and acetonitrile in different ratio as mobile phase in a ternary linear gradient mode. The calibration graphs plotted with five different concentrations of each component were linear with a regression coefficient R2 >0.999. The limit of detection and limit of quantitation were estimated for all the components. Effect on analytical responses by small and deliberate variation of critical factors was examined by robustness testing with Design of Experiment employing Central Composite Design and established that this method was robust. The method was then validated for linearity, precision, accuracy, specificity and demonstrated to be applicable to the determination of the ingredients in commercial sample of essential oil. PMID:24799735

  10. Simultaneous HPLC determination of 22 components of essential oils; method robustness with experimental design.

    PubMed

    Porel, A; Sanyal, Y; Kundu, A

    2014-01-01

    The aim of the present study was the development and validation of a simple, precise and specific reversed phase HPLC method for the simultaneous determination of 22 components present in different essential oils namely cinnamon bark oil, caraway oil and cardamom fruit oil. The chromatographic separation of all the components was achieved on Wakosil-II C18 column with mixture of 30 mM ammonium acetate buffer (pH 4.7), methanol and acetonitrile in different ratio as mobile phase in a ternary linear gradient mode. The calibration graphs plotted with five different concentrations of each component were linear with a regression coefficient R(2) >0.999. The limit of detection and limit of quantitation were estimated for all the components. Effect on analytical responses by small and deliberate variation of critical factors was examined by robustness testing with Design of Experiment employing Central Composite Design and established that this method was robust. The method was then validated for linearity, precision, accuracy, specificity and demonstrated to be applicable to the determination of the ingredients in commercial sample of essential oil. PMID:24799735

  11. A validated stability-indicating HPLC method for the determination of PEGylated puerarin in aqueous solutions.

    PubMed

    Liu, Xinyi; Yu, Boyang; Wang, Naijie; Zhang, Bei; Du, Feng; He, Cheng; Ye, Zuguang

    2010-08-01

    The aim of this study was to develop a validated specific stability-indicating HPLC method for the quantitative determination of PEGylated puerarin (PEG-PUE) in aqueous solutions. The method was validated by subjecting PEG-PUE to forced degradation under stress conditions of acid, alkali, water hydrolysis, and oxidation. Both PEG-PUE and puerarin (PUE) were simultaneously determined and separated on CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1.0 mL min(-1) and detection wavelength was set at 250 nm. Both calibration curves showed good linear regression (r> or =0.9998) within test ranges. The LOD and LOQ of PEG-PUE were determined to be 3 and 9 microg mL(-1) respectively. Degradation of PEG-PUE followed pseudo-first-order kinetics with t(1/2) of 59 min at pH 9.0 and 17.79 h at pH 7.4. However, at pH 5.0 and 2.0, there was no significant degradation of PEG-PUE over time. In conclusion, the method was observed to have the necessary specificity, precision, and accuracy, and to be suitable for quantity monitoring the degradation process of PEG-PUE during stability studies. The degradation studies may give insight into useful information for formulation development of PEG-PUE.

  12. [Simultaneous determination of neotame, alitame and aspartame in foods by HPLC].

    PubMed

    Matsumoto, Hiroko; Hirata, Keiko; Sakamaki, Narue; Hagino, Kayo; Ushiyama, Hirofumi

    2008-02-01

    Simultaneous determination of three artificial sweeteners, neotame (NE), alitame (AL) and aspartame (APM) in various foods by high-performance liquid chromatography (HPLC) was developed. Chopped or homogenized samples were packed into cellulose tubing with 0.01 mol/L hydrochloric acid containing 10% sodium chloride, and dialyzed against 0.01 mol/L hydrochloric acid for 24-48 hours. The dialyzate was passed through an Oasis MCX cartridge, and the cartridge was washed with water and methanol. Then the three sweeteners were eluted from the cartridge with a mixture of 0.5 mol/L ammonium chloride-acetonitrile (3 : 2). The sweeteners were separated on a Cosmosil 5C18-AR column using a gradient mode with a mobile phase of 0.01 mol/L phosphate buffer (pH 4.0)-acetonitrile and were detected at 210 nm. The recoveries of NE, AL and APM from 8 kinds of foods spiked with 10 and 100 microg/g were 86-100% and 89-104%, respectively. The detection limits of NE, AL and APM were 1 microg/g in samples. Furthermore, the three sweeteners were successfully identified by using liquid chromatography with tandem mass spectrometry.

  13. Simultaneous determination of grepafloxacin, ciprofloxacin, and theophylline in human plasma and urine by HPLC.

    PubMed

    Kamberi, M; Hajime, N; Kamberi, P; Uemura, N; Nakamura, K; Nakano, S

    1999-06-01

    A specific and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of grepafloxacin, ciprofloxacin, and theophylline in human plasma and urine. This assay allows these drugs to elute and be resolved in a single chromatogram at 280 nm, using a linear gradient. The procedure involves liquid-liquid extraction. Separation was achieved on a C18 reversed-phase column. The quantification limits were 0.05 mg/L in plasma and 0.5 mg/L in urine for grepafloxacin and ciprofloxacin and 0.5 mg/L in plasma and urine for theophylline. Standard curves were linear (correlation coefficients >0.999) over the ranges 0.05 to 5 mg/L for grepafloxacin and ciprofloxacin in plasma, from 0.5 to 20 mg/L for theophylline in plasma, and from 0.5 to 500 mg/L for the three drugs in urine. The coefficients of variation for the three drugs were less than 10% for within- and between-day analyses. The recoveries averaged 94.5% for theophylline, 93% for ciprofloxacin, 93.7% for grepafloxacin, and 95.1% for the internal standard (IS). The assay can be used for pharmacokinetic studies of these drugs, to investigate the interaction of grepafloxacin and ciprofloxacin with theophylline, or for routine simultaneous monitoring of theophylline, grepafloxacin, and ciprofloxacin.

  14. HPLC-ICP atomic emission spectrometry in environmental analysis

    SciTech Connect

    Frame, E.M.S.

    1996-10-01

    Trace concentrations of many compounds are difficult to determine directly by traditional chromatographic techniques. Organosilicon compounds, for example, have no chromophoric groups. Size exclusion (SEC) and high performance liquid chromatography (HPLC) can separate high and low molecular weight organosilicon compounds, but routinely employ detectors with insufficient sensitivity for trace concentrations in environmental samples. Elemental analysis methods such as inductively coupled plasma optical emission spectrometry (ICP) provide excellent element sensitivity but no speciation information. Interfacing of a chromatographic method with ICP has been shown to provide quantitative determination of trace compounds while retaining the chemical speciation information. This work will describe the power of the HPLC-ICP combination for trace compounds in environmental samples to identify and determine ppm levels of organometallics.

  15. Prototype sphere-on-sphere silica particles for the separation of large biomolecules.

    PubMed

    Fekete, Szabolcs; Rodriguez-Aller, Marta; Cusumano, Alessandra; Hayes, Richard; Zhang, Haifei; Edge, Tony; Veuthey, Jean-Luc; Guillarme, Davy

    2016-01-29

    The goal of this study was to evaluate the possibilities offered by a prototype HPLC column packed with ∼2.5μm narrow size distribution sphere-on-sphere (SOS) silica particles bonded with C4 alkyl chains, for the analytical characterization of large biomolecules. The kinetic performance of this material was evaluated in both isocratic and gradient modes using various model analytes. The data were compared to those obtained on other widepore state-of-the-art fully core-shell and fully porous materials commonly employed to separate proteins moreover to a reference 5μm wide pore material that is still often used in QC labs. In isocratic mode, minimum reduced plate height values of hmin=2.6, 3.3 and 3.3 were observed on butylparaben, decapeptide and glucagon, respectively. In gradient elution mode, the SOS column performs very high efficiency when working with fast gradients. This prototype column was also comparable (and sometimes superior) to other widepore stationary phases, whatever the gradient time and flow rate, when analyzing the largest model protein, namely BSA. These benefits may be attributed to the SOS particle morphology, minimizing the intra-particle mass transfer resistance. Finally, the SOS column was also applied for the analytical characterization of commercial monoclonal antibody (mAb) and antibody-drug conjugate (ADC) samples. With these classes of proteins, the performance of SOS column was similar to the best widepore stationary phases available on the market.

  16. Prototype sphere-on-sphere silica particles for the separation of large biomolecules.

    PubMed

    Fekete, Szabolcs; Rodriguez-Aller, Marta; Cusumano, Alessandra; Hayes, Richard; Zhang, Haifei; Edge, Tony; Veuthey, Jean-Luc; Guillarme, Davy

    2016-01-29

    The goal of this study was to evaluate the possibilities offered by a prototype HPLC column packed with ∼2.5μm narrow size distribution sphere-on-sphere (SOS) silica particles bonded with C4 alkyl chains, for the analytical characterization of large biomolecules. The kinetic performance of this material was evaluated in both isocratic and gradient modes using various model analytes. The data were compared to those obtained on other widepore state-of-the-art fully core-shell and fully porous materials commonly employed to separate proteins moreover to a reference 5μm wide pore material that is still often used in QC labs. In isocratic mode, minimum reduced plate height values of hmin=2.6, 3.3 and 3.3 were observed on butylparaben, decapeptide and glucagon, respectively. In gradient elution mode, the SOS column performs very high efficiency when working with fast gradients. This prototype column was also comparable (and sometimes superior) to other widepore stationary phases, whatever the gradient time and flow rate, when analyzing the largest model protein, namely BSA. These benefits may be attributed to the SOS particle morphology, minimizing the intra-particle mass transfer resistance. Finally, the SOS column was also applied for the analytical characterization of commercial monoclonal antibody (mAb) and antibody-drug conjugate (ADC) samples. With these classes of proteins, the performance of SOS column was similar to the best widepore stationary phases available on the market. PMID:26755414

  17. Analysis of flavonoids and iridoids in Vitex negundo by HPLC-PDA and method validation.

    PubMed

    Roy, Somendu K; Bairwa, Khemraj; Grover, Jagdeep; Srivastava, Amit; Jachak, Sanjay M

    2013-09-01

    The leaves of Vitex negundo have been reported to contain various bioactive constituents including iridoids and flavonoids. This is the first report on the simultaneous determination of iridoids and flavonoids by HPLC in three different samples of V. negundo leaves collected from three regions of India. Separation of iridoids and flavonoids was accomplished by HPLC and further elaborated for their quantification in V. negundo leaves using a C-18 column with detection at 254 and 330 nm, respectively. The developed HPLC method showed good linearity (r2 > or = 0.999), high precision (RSD < 5%) and a good recovery (99.3-103.0%) of the compounds. All the validation parameters of the developed HPLC were found to be within the permissible limits according to the ICH guidelines. The developed method was robust, accurate and reliable for the quality control of V. negundo leaves.

  18. HPLC-MTT assay: anticancer activity of aqueous garlic extract is from allicin.

    PubMed

    Lee, Jenny; Gupta, Shalini; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Bao-Shiang

    2013-05-15

    A strategy using reversed-phase high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrometry (MS), nuclear magnetic resonance (NMR), chemical synthesis, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay to identify allicin as the active anticancer compound in aqueous garlic extract (AGE) is described. Changing the pH of AGE from 7.0 to 5.0 eliminated interfering molecules and enabled a clean HPLC separation of the constituents in AGE. MTT assay of the HPLC fractions identified an active fraction. Further analysis by TLC, MS, and NMR verified the active HPLC fraction as allicin. Chemically synthesized allicin was used to provide further confirmation. The results clearly identify the active compound in AGE as allicin.

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

    PubMed

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

    2012-01-27

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

  20. High gradient directional solidification furnace

    NASA Technical Reports Server (NTRS)

    Aldrich, B. R.; Whitt, W. D. (Inventor)

    1985-01-01

    A high gradient directional solidification furnace is disclosed which includes eight thermal zones throughout the length of the furnace. In the hot end of the furnace, furnace elements provide desired temperatures. These elements include Nichrome wire received in a grooved tube which is encapsulated y an outer alumina core. A booster heater is provided in the hot end of the furnace which includes toroidal tungsten/rhenium wire which has a capacity to put heat quickly into the furnace. An adiabatic zone is provided by an insulation barrier to separate the hot end of the furnace from the cold end. The old end of the furnace is defined by additional heating elements. A heat transfer plate provides a means by which heat may be extracted from the furnace and conducted away through liquid cooled jackets. By varying the input of heat via the booster heater and output of heat via the heat transfer plate, a desired thermal gradient profile may be provided.

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

    NASA Technical Reports Server (NTRS)

    Morales, W.

    1986-01-01

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

  2. [HPLC determination of troxerutin in plasma and urine following oral administration in man].

    PubMed

    Dittrich, P; Ostrowski, J; Beubler, E; Schraven, E; Kukovetz, W

    1985-01-01

    A high-performance liquid chromatographic method (HPLC) is described for the analysis of 3',4',7-tri-O-(beta-hydroxy-ethyl)-rutoside (troxerutin) in human plasma and urine. After separation of interfering substances on XAD-2 trihydroxyethylrutoside is converted to tetrahydroxyethylrutoside by 2-chlorethanol in alkaline medium. After HPLC-separation tetrahydroxyethylrutoside is quantified by fluorescence detection. The pharmacokinetics of troxerutin were measured in plasma after oral administration to man. The relative bioavailability of the drug from Venelbin was 97.8 +/- 37.1% compared to an aqueous standard solution.

  3. A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

    PubMed

    Moghbel, Nahid; Ryu, BoMi; Steadman, Kathryn J

    2015-08-01

    A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD <2%. Limit of detection was 1.6μg/mL for nicotine and below 1μg/mL for all other alkaloids. The limit of quantification was 2.8 and 4.8μg/mL for nornicotine and nicotine respectively, and below 2μg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.

  4. The use of pH-gradient ion-exchange chromatography to separate sheep liver cytoplasmic aldehyde dehydrogenase from mitochondrial enzyme contamination, and observations on the interaction between the pure cytoplasmic enzyme and disulfiram.

    PubMed Central

    Dickinson, F M; Hart, G J; Kitson, T M

    1981-01-01

    1. Sheep liver cytoplasmic aldehyde dehydrogenase can be purified from contamination with the mitochondrial form of the enzyme by pH-gradient ion-exchange chromatography. The method is simple, reproducible and efficient. 2. The purified cytoplasmic enzyme retains about 2% of its original activity in the presence of a large excess of disulfiram. This suggests that the disulfiram-reactive thiol groups are not essential for covalent interaction with the aldehyde substrate during catalysis, as has sometimes been suggested. 3. Between 1.5 and 2.0 molecules of disulfiram per tetrameric enzyme molecule account for the observed loss of activity, suggesting that the enzyme may have only two functional active sites. 4. Experiments show that disulfiram-modified enzyme retains the ability to bind NAD+ and NADH. PMID:7340819

  5. TLC and HPLC characteristics of desacetylmatricarin, leucodin, achillin and their 8alpha-angeloxy-derivatives.

    PubMed

    Glasl, S; Mucaji, P; Werner, I; Jurenitsch, J

    2003-07-01

    Five guaianolides, including two pairs of isomers, from a Hungarian taxon of the Achillea millefolium group were characterized analytically. Different chromatographic systems on TLC and HPLC were developed for the analysis of these compounds. TLC of leucodin, 8alpha-angeloxy-leucodin, achillin, 8alpha-angeloxy-achillin and desacetylmatricarin was performed on silica gel using dichloromethaneacetone and cyclohexane-ethylacetate mixtures as mobile phases. HPLC on stationary phases as LiChrospher RP2, LiChrospher RP8, LiChrospher RP18e, Hypersil BDS C18 and Aquasil C18 required isocratic and gradient systems with different methanol-water mixtures as mobile phases. The presented RF values and retention times allow the identification of the respective 2-oxo-guaianolides which are marker substances for certain non-proazulene containing species. Their TLC and HPLC fingerprints are compared to those of proazulene containing species and are relevant for quality control.

  6. Moving thermal gradients in gas chromatography.

    PubMed

    Tolley, H Dennis; Tolley, Samuel E; Wang, Anzi; Lee, Milton L

    2014-12-29

    This paper examines the separation effects of a moving thermal gradient on a chromatographic column in gas chromatography. This movement of the gradient has a focusing effect on the analyte bands, limiting band broadening in the column. Here we examine the relationship between the slope of this gradient, the velocity of the gradient and the resulting band width. Additionally we examine how transport of analytes along the column at their analyte specific constant temperatures, determined by the gradient slope and velocity, affects resolution. This examination is based primarily on a theoretical model of partitioning and transport of analyte under low concentration conditions. Preliminary predictions indicate that analytes reach near constant temperatures, relative positions and resolutions in less than 100cm of column transport. Use of longer columns produces very little improvement in resolution for any fixed slope. Properties of the thermal gradient determine a fixed solute band width for each analyte. These widths are nearly reached within the first 40-70cm, after which little broadening or narrowing of the bands occur. The focusing effect of the thermal gradient corrects for broad injections, reduces effects of irregular stationary phase coatings and can be used with short columns for fast analysis. Thermal gradient gas chromatographic instrumentation was constructed and used to illustrate some characteristics predicted from the theoretical results.

  7. Separation of Peptides on HALO 2-Micron Particles.

    PubMed

    Mant, Colin T; Hodges, Robert S

    2016-01-01

    Reversed-phase high-performance liquid chromatography (RP-HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP-HPLC stationary-phase materials with enhanced separation efficiency. HALO packing materials began the revolution in "core-shell" technology with the advantages of faster separations, higher resolution and peak capacity, high temperature stability, and rugged reliable performance compared to traditional HPLC and UHPLC. These materials are characterized by a solid core surrounded by a thin layer of porous material, and represent a technology for the future with continuing refinements. Such refinements are aided via the use of designed synthetic peptide standards during stationary-phase development. Concomitantly, such standards also enable the researcher to monitor RP-HPLC column performance and develop optimized separation protocols for peptides from a wide array of sources. © 2016 by John Wiley & Sons, Inc. PMID:27479502

  8. Simultaneous quantification of five steroid saponins from Dioscorea zingiberensis C.H. Wright in rat plasma by HPLC-MS/MS and its application to the pharmacokinetic studies.

    PubMed

    Zhang, Xinxin; Li, Jing; Ito, Yoichiro; Sun, Wenji

    2015-01-01

    A simple, reliable and sensitive high-performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) was established for simultaneous analyses of the following 5 steroid saponins in rat plasma after the single dose administration of total steroid saponins extracted from the rhizome of Dioscorea zingiberensis C.H. Wright for the first time. Protodioscin, huangjiangsu A, zingiberensis new saponin, dioscin, and gracillin were quantified using ginsenoside Rb1 as the internal standard (IS). The plasma samples were pretreated by a single step acetonitrile-mediated protein precipitation. The chromatographic separation was performed on an Inersil ODS-3 C18 column (250mm×4.6mm, 5μm) with the mobile phase composed of acetonitrile and water containing 0.1% formic acid under a gradient elution mode at 0.2mLmin(-1) using a microsplit after the eluent from the HPLC apparatus. The quantification was accomplished on a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) in the positive ionization mode. The above five analytes were stable under sample storage and preparation conditions applied in the present study. The linearity, precision, accuracy, and recoveries of the analysis confirmed the requirements for quality-control purposes. After validation, this proposed method was successfully adopted to investigate the pharmacokinetic parameters of these five analytes.

  9. HPLC-ESI-MS/MS analysis and pharmacokinetics of luteoloside, a potential anticarcinogenic component isolated from Lonicera japonica, in beagle dogs.

    PubMed

    Qiu, Feng; Li, Zhaoxia; He, Lang; Wang, Dan

    2013-03-01

    Luteoloside is a potential anticarcinogenic component isolated from Lonicera japonica, a traditional Chinese medicine (TCM). This study details the development and validation of a sensitive and accurate HPLC-ESI-MS/MS method for the quantification of luteoloside in dog plasma. Sample pretreatment includes simple protein precipitation using methanol-acetonitrile (1:1, v/v). A Phenomenex Gemini C(18) column (2.0 × 50 mm, i.d., 3.5 µm) was used to separate luteoloside and internal standard by gradient mode with mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid at a flow rate of 0.40 mL/min with a column temperature of 25°C. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode. The calibration curves were linear (R > 0.995) over the concentration range 1.0-2000 ng/mL and the lower limit of quantification was 1.0 ng/mL. The intra-day and inter-day precisions (RSD) were all <15%, accuracies (RE) were within the range of ±15%, and recoveries were between 85.0 and 115%. The validated HPLC-ESI-MS/MS method was successfully applied to determine plasma concentrations of luteoloside after intravenous administration of luteoloside at a dose level of 20 mg/kg.

  10. Screening of preservatives by HPLC-PDA-ESI/MS: A focus on both allowed and recently forbidden compounds in the new EU cosmetics regulation.

    PubMed

    Lecce, Raffaele; Regazzoni, Luca; Mustazza, Carlo; Incarnato, Giampaolo; Porrà, Rita; Panusa, Alessia

    2016-06-01

    Commission regulation (EU) No 358/2014 amending the new regulation (EC) No 1223/2009 on cosmetics has prohibited the use of isopropyl-, isobutyl-, phenyl-, benzyl- and pentylparaben. Furthermore, Commission regulation (EU) No 1004/2014 has lowered the maximum permitted concentration of butyl- and propylparaben in cosmetics and it has also banned them in leave-on products designed for application on the nappy area of children under three years of age. A HPLC-PDA-ESI/MS method has been developed herein for the detection of seventeen preservatives, both the most utilised and the recently forbidden by the new EU regulations. The separation of these compounds, including benzoic acid and its derivatives in a 1.10 - 3.04 log Pow range, has been performed with a gradient elution on a Symmetry(®) C18 column (250×4.6mm i.d., particle size 5μm) with water and acetonitrile (0.1% formic acid) as mobile phase. Quantification has been carried out by HPLC-PDA. The method has been validated and successfully applied to the analysis of a large number of cosmetics with different functions like rinse-off and leave-on, or composition like skin, hair, face and oral products. PMID:27055177

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2013-12-01

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

  13. Isolation, Chemical Fingerprinting and Simultaneous Quantification of Four Compounds from Tanacetum gracile Using a Validated HPLC-ESI-QTOF-Mass Spectrometry Method.

    PubMed

    Sharma, Neha; Kumar, Chetan; Dutt, Prabhu; Gupta, Suphla; Satti, Naresh K; Chandra, Suresh; Kitchlu, Surinder; Paul, Satya; Vishwakarma, Ram A; Verma, Mahendra K

    2016-01-01

    The present study was conducted to carry out the phytochemical investigation of Tanacetum gracile Hook. f. & Thomson and to develop a method for the simultaneous quantification of the isolated compounds in the extracts ofT. gracile growing in different locations. Cluster analysis rectangular similarity matrix was performed to understand the chemical fingerprinting variations in the extracts. High-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight-mass spectrometry (HPLC-ESI-QTOF-MS) was used to quantify four bioactive compounds, and separation of the compounds was achieved on a reverse-phase C8 column using a mobile phase of acetonitrile: 0.1% formic acid in water with a gradient elution by maintaining the flow rate of 300 μL/min. The QTOF-MS was operated using the electro-spray ionization technique with the positive ion polarity mode. The calibration curves of four marker compounds were linear over the concentration range of 3.12-100 ng/µL (R(2)> 0.996). A specific, accurate and precise HPLC-ESI-QTOF-MS method was optimized for the determination of kaempferol, ketoplenolide, tetramethoxyflavone and artemetin both individually and simultaneously. Quantification of these chemical markers in different extracts was carried out using this validated method. Kaempferol was isolated for the first time from T. gracile. PMID:26951542

  14. Separation and identification of polyphenols in apple pomace by high-speed counter-current chromatography and high-performance liquid chromatography coupled with mass spectrometry.

    PubMed

    Cao, Xueli; Wang, Cong; Pei, Hairun; Sun, Baoguo

    2009-05-01

    Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane-ethyl acetate-1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace.

  15. Stereo transparency and the disparity gradient limit

    NASA Technical Reports Server (NTRS)

    McKee, Suzanne P.; Verghese, Preeti

    2002-01-01

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

  16. Surface confined ionic liquid as a stationary phase for HPLC

    SciTech Connect

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

    2006-01-01

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

  17. Simultaneous quantification of eleven bioactive components of male flowers of Eucommia ulmoides oliver by HPLC and their quality evaluation by chemical fingerprint analysis with hierarchical clustering analysis

    PubMed Central

    Ding, Yanxia; Dou, Deqiang; Guo, Yangjing; Li, Qin

    2014-01-01

    Background: Eucommia ulmoides Oliv (EU), a dioecious perennial angiosperm, is one of the oldest tonics in Chinese traditional medicine. The tea of male flowers of EU has been become popularities and seen as aspirational health care tea in China. There were no enough marks and effective method to control the quality of male flowers of EU. Objective: A simple and efficient HPLC method was developed for the simultaneous determination of 11 bioactive compounds (4 iridoids, 1 phenylpropanoid, 6 flavonoids). HPLC chromatographic fingerprint and hierarchical cluster analysis were used to evaluate and classify the samples of male flowers of EU which came from different locations in China. Materials and Methods: Samples were separated on a Thermal hypersil gold column (250 mm × 4.6 mm, 5 μm) and detected by an ultraviolet detector. The UV wavelength was set at 206, 236, and 206 nm. Mobile phase consisted of methanol (B) and phosphoric acid-water (0.5%) (C) using a gradient elution. Analytes were performed at 25°C with a flow rate of 1.0 mL/min. Results: In quantitative analysis, the eleven components showed good regression (r2 > 0.9996) within linear ranges, and their recoveries were in the range of 98.65-102.31%. In the chromatographic fingerprint, 16 peaks were selected as the characteristic peaks to assess the similarities of different samples. Hierarchical cluster analysis (HCA) was also applied to differentiate the samples based on the area of all the common peaks. The samples which had higher similarity in HPLC fingerprint were classified as a cluster. Conclusion: This study will provide methodological reference for the quality control and sample classification of male flowers of E. ulmoides. PMID:25422543

  18. Simultaneous and group determination methods for designated substances by HPLC with multi-channel electrochemical detection and their application to real samples.

    PubMed

    Min, Jun Zhe; Yamashita, Kazuhide; Toyo'oka, Toshimasa; Inagaki, Shinsuke; Higashi, Tatsuya; Kikura-Hanajiri, Ruri; Goda, Yukihiro

    2010-12-01

    Many psychotropic substances are illegally available on the streets and/or via the Internet. This wide distribution has become a serious social problem. To control this problem, many substances have been controlled as 'designated substances' (Shitei-Yakubutsu) in Japan since April 2007 by the Pharmaceutical Affairs Law, including tryptamines, phenethylamines and piperazines. In the present study, simultaneous determination methods using HPLC with multi-channel electrochemical detection (MECD) were developed for the designated substances. The proposed methods utilizing online electrochemical oxidation are the first report on the simultaneous determination of various designated substances. The methods involve direct determination and require no complicated pretreatments such as fluorescence labeling. The designated substances were separated by reversed-phase chromatography using a TSK-gel ODS-100V (4.6 × 250 mm, i.d., 3 µm) and gradient elution by a mixture of potassium phosphate buffer, methanol and acetonitrile. The total separation of 31 designated substances was successfully performed but required long chromatographic run times. Thus, the designated substances were divided into three groups: (1) tryptamines, (2) phenethylamines and (3) piperazines and others. They were then analyzed by HPLC-MECD as another separation method. The suitable applied voltages for each designated substance were determined based upon the hydrodynamic voltammogram. The limits of detection (signal-to-noise ratio of 3) of the designated substances for the most suitable voltages were in the range of 17.1 pg (5-MeO-MIPT) to 117 ng (indan-2-amine). The calibration curves based on the peak heights were linearly related to the amounts of the designated substances (R(2) > 0.999). Good accuracy and precision by intra-day assay and inter-day assay were also obtained using the present procedures. The proposed methods were applied to the analyses of the designated substance in several real

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

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  20. Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

    PubMed Central

    Slocum, Robert D.; Flores, Hector E.; Galston, Arthur W.; Weinstein, Leonard H.

    1989-01-01

    The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucus carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues. Images Figure 4 Figure 5 PMID:11537449

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

    PubMed

    Zhou, Jue; Qu, Fan

    2011-01-01

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

  2. Analysis of a simulated heroin distribution chain by HPLC.

    PubMed

    Zelkowicz, Avraham; Magora, Amir; Ravreby, Mark D; Levy, Rina

    2005-07-01

    A heroin distribution chain was simulated by taking three different seizures and preparing four additional samples from each seizure by adding a paracetamol-caffeine mixture in varying amounts, resulting in three different batches each composed of five samples. All of the samples from the three batches were analyzed using HPLC with a UV-PDA detector at a wavelength of 230 nm. The area ratio of various opium alkaloids, acetylation products and components were compared. From the results of the UV area ratios, the fifteen samples could readily be separated into three batches of five samples, with each batch of five samples having a common origin.

  3. Determination of Sinomenine in Cubosome Nanoparticles by HPLC Technique

    PubMed Central

    Zhou, Yanfang; Guo, Chunlian; Chen, Hongying; Zhang, Yudai; Peng, Xinsheng; Zhu, Ping

    2015-01-01

    We applied HPLC technique to quantitatively analyze sinomenine in cubosome nanoparticles. The chromatographic method was performed by using an isocratic system. The mobile phase was composed of methanol-PBS(pH6.8)-triethylamine (50 : 50 : 0.1%) with a flow rate of 1 mL/min; the detection wavelength was at 265 nm. Sinomenine can be successfully separated with good linearity (the regression equation is A = 10835C + 1058; R2 = 1.0) and perfect recovery (102.2%). The chromatograph technique was proper for quality control of sinomenine in cubosome nanoparticles. PMID:25734024

  4. A Validated HPLC Method for Simultaneous Determination of Caffeoyl Phenylethanoid Glucosides and Flavone 8-C-glycosides in Haberlea rhodopensis.

    PubMed

    Zheleva-Dimitrova, Dimitrina; Nedialkov, Paraskev; Giresser, Ulrich

    2016-06-01

    A HPLC-UV method for analysis of the main compounds: caffeoyl phenylethanoid glucosides myconoside (1) and paucifloside (2) and flavone 8-C-glycosides: hispidulin 8-C-β-galactopyranoside (3), hispidulin 8-C-(2"-O-syringoyl-β-glucopyranoside) (4), hispidulin 8-C-(6-O-acetyl-β-glucopyranoside) (5) and hispidulin 8-C-(6-O-acetyl-2"-O-syringoyl--glucopyranoside) (6) in Haberlea rhodopensis leaves was developed and validated. Compound 3 was isolated for the first time from the title species. Ultrasound extraction with 80% methanol at room temperature allowed a good recovery of analytes (from 87.2 % for 1 to 109.8 % for 3) and the precision of the entire procedure was between 1.6% and 6.9%. The subsequent HPLC separation and quantification was achieved using a Hypersil ODS C18 column and UV detection at 280 nm. The mobile phase comprised methanol and 0.1 % o-phosphoric acid, and gradient elution mode was applied. The detection limits ranged from 0.042 μg/mL (6) to 0.18 μg/mL (5). The total amount in leaves of the assayed phenolic compounds was 374.2 mg/g. Myconoside was found to be the dominant compound in H. rhodopensis extract (332.2 ± 0.7 mg/g dw) and reached up to 88.8% of the analyzed mixture in leaves, while the total content of flavone C-glycosides was 17.1 mg/g dw. PMID:27534117

  5. Simultaneous determination of phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC-DAD-ESI-MS.

    PubMed

    Inbaraj, B Stephen; Lu, H; Kao, T H; Chen, B H

    2010-02-01

    A high-performance liquid chromatography-diode array detection-mass spectrometry method with electrospray ionization mode (HPLC-DAD-ESI-MS) was developed for simultaneous determination of phenolic acids and flavonoids in fruits of Lycium barbarum Linnaeus, a widely used traditional Chinese herb possessing vital biological activity. Both phenolic acids and flavonoids were extracted with 50% ethanol and purified using a polymeric solid phase extraction cartridge followed by HPLC-DAD-ESI-MS analysis. By employing a Vydac C18 column, a total of 52 phenolic acids and flavonoids were separated within 70min using a gradient mobile phase of 0.5% (v/v) formic acid in water and acetonitrile-water (94:6, v/v) with flow rate at 1mL/min, column temperature at 30 degrees C and detection wavelength at 280nm. Of 52 compounds, 15 phenolic acids and flavonoids were positively identified based on both absorption and mass spectra, with the remaining 37 tentatively identified by comparison of absorption spectra with reported values in the literature. Internal standards 3-hydroxybenzoic acid and hesperidin were used for quantitation of phenolic acids and flavonoids, respectively. Among the 15 positively identified compounds, quercetin-rhamno-di-hexoside was present in largest mass fraction (438.6microg/g), followed by quercetin-3-O-rutinoside (281.3microg/g), dicaffeoylquinic acid isomers (250.1microg/g), chlorogenic acid (237.0microg/g), quercetin-di-(rhamnohexoside) (117.5microg/g), quercetin-di-(rhamno)-hexoside (116.8mug/g), kaempferol-3-O-rutinoside (97.7microg/g), isorhamnetin-3-O-rutinoside (72.1microg/g), p-coumaric acid (64.0microg/g), caffeic acid (23.7microg/g) and vanillic acid (22.8microg/g). PMID:19819093

  6. Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction.

    PubMed

    Sun, Han-Wen; Wang, Feng-Chi; Ai, Lian-Feng

    2007-10-01

    A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C(18) bonded silica column with a deionized water-methanol-acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 microg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1-20 microg/kg were in the range of 71.4-99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 microg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 microg/kg were in the range of 6.2-13.9% and 4.0-8.7%, respectively. The intra-day precision (n=5) for nitroimidazoles residues in chicken spiked at 20 microg/kg is 6.9%, and the inter-day precision for 5 days (n=25) is 11%. The method is capable of identifying nitroimidazole residues at > or =0.7 microg/kg levels and was applied in the determination of nitroimidazole residues in meat sample. PMID:17719280

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

    PubMed

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

    2008-10-19

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

  8. Simultaneous analysis of several antiretroviral nucleosides in rat-plasma by high-performance liquid chromatography with UV using acetic acid/hydroxylamine buffer Test of this new volatile medium-pH for HPLC-ESI-MS/MS.

    PubMed

    Bezy, Vincent; Morin, Philippe; Couerbe, Philippe; Leleu, Ghislaine; Agrofoglio, Luigi

    2005-07-25

    Zalcitabine (ddC), lamivudine (3TC), didanosine (ddI), stavudine (d4T), carbovir (CBV), zidovudine (AZT), tenofovir (PMPA) and its administrated form (tenofovir diisoproxyl fumarate, TDF), are nucleosides currently approved in HIV therapy. To facilitate pharmacokinetics studies, a specific reversed-phase high-performance liquid chromatography (HPLC) method was developed for their analysis in rat plasma. The method involved a quantitative recovery of these drugs from rat plasma by solid-phase extraction on Oasis HLB Waters cartridges followed by optimised HPLC separation on an Atlantis dC18 column with acetic acid-hydroxylamine buffer (ionic strength 5mM, pH 7)-acetonitrile elution gradient. Quantitation was performed by HPLC/UV at 260 nm. Linear calibration curves were obtained within a 30-10,000 ng/mL plasma concentration range. Correlation coefficients (r2) greater than 0.992 were obtained by least-squares regression and limits of quantification were in 30-90 ng/mL concentration range. Quantitative parameters (accuracy, intra-day repeatability and inter-day reproducibility) yielded satisfactory results. Finally, a new buffer, obtained with acetic acid and hydroxylamine, has been tested in HPLC/ESI-MS/MS and appears to be an efficient volatile buffer in the medium 5-7 pH range. Indeed, at pH 7 and low ionic strength (5 mM), its buffer capacity is one hundred times higher to that obtained for the usual acetic acid/ammonia buffer.

  9. Investigation of Gravity Lanthanide Separation Chemistry

    SciTech Connect

    Payne, Rosara F.; Schulte, Shannon M.; Douglas, Matthew; Friese, Judah I.; Farmer, Orville T.; Finn, Erin C.

    2011-03-01

    Lanthanides are common fission products and the ability to separate and quantify these elements is critical to rapid radiochemistry applications. Published lanthanide separations using Eichrom Ln Spec resin utilize an HCl gradient. Here it is shown that the efficacy and resolution of the separation is improved when a nitric acid gradient is used instead. The described method allows parallel processing of many samples in 1.5 hours followed by 60 minute counting for quantification of 9 isotopes of 7 lanthanide elements.

  10. Determination of capsinoids by HPLC-DAD in capsicum species.

    PubMed

    Singh, Satyavan; Jarret, Robert; Russo, Vincent; Majetich, George; Shimkus, Joel; Bushway, Rodney; Perkins, Brian

    2009-05-13

    Capsicum fruits contain a newly discovered phytochemical called capsinoids. Because little is known about the quantities of these compounds in both sweet and pungent pepper fruits, a high-performance liquid chromatography (HPLC) method was developed to identify and quantify the capsinoids (naturally present E-capsiate and dihydrocapsiate) utilizing fruit obtained from a variety of Capsicum spp. in the U.S. Department of Agriculture's Capsicum germplasm collection. Capsinoids were extracted with acetonitrile, filtered, and analyzed using an HPLC system equipped with a C(18) monolithic column, gradient pump, and diode array detector. The elution solvents were acetonitrile and water (60:40) with an isocratic flow rate of 1.0 mL/min. Forty-nine samples representing distinct morphotypes of four cultivated species ( C. annuum var. annuum, C. annuum var. glabriusculum, C. baccatum , C. chinense , and C. frutescens ) contained detectable levels (11-369 microg/g) of E-capsiate quantified at a wavelength of 280 nm. Nine of the E-capsiate-containing samples also had dihydrocapsiate (18-86 micro/g). Gas chromatography with a mass spectrometry detector (GC-MS) confirmed the presence of these compounds in the Capsicum spp.

  11. [Determination of furbenicillin sodium and its related substances by HPLC].

    PubMed

    Chang, Yan; Tian, Ye; Ma, Yue; Hu, Chang-qin

    2015-12-01

    Furbenicillin is a broad-spectrum semisynthetic penicillin with strong antibacterial activity against Gram-negative bacteria. Furbenicillin sodium is determined by volumetric method in current criteria. However, the criteria does not contain an assay of related substances of furbenicillin sodium. In this study, we established a method for detection and analysis of furbenicillin sodium and its related substances by HPLC. The analysis was performed with a C18 column under a gradient elution, the detection wavelength was 225 nm, and the column temperature was 35 degrees C. The reliability and accuracy of established method was validated in this study. Pure samples of furbenicillin sodium and its related substances were prepared. The structures, biological activities, and chromatographic retention behaviors of furbenicillin sodium and its related substances were identified using NMR, CLSI agar dilution method, and HPLC. All results in the current study provide ample evidence that this method is able to determine the reasonable limits in the quality-control protocol for furbenicillin sodium. PMID:27169288

  12. Use of the bromine isotope ratio in HPLC-ICP-MS and HPLC-ESI-MS analysis of a new drug in development.

    PubMed

    Cuyckens, Filip; Balcaen, Lieve I L; De Wolf, Kenny; De Samber, Bjorn; Van Looveren, Cis; Hurkmans, Rob; Vanhaecke, Frank

    2008-04-01

    A combination of inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization mass spectrometry (ESI-MS) was deployed for the metabolite profiling and metabolite identification of a new antituberculosis compound (R207910, also known as TMC207) that is currently in drug development. R207910 contains one bromine atom, allowing the detection by ICP-MS. Fluctuations in the Br sensitivity caused by the HPLC gradient were counteracted by the use of species-unspecific isotope dilution. In order to evaluate the method developed, the results obtained were compared with those acquired via radioactivity detection. HPLC-ESI-MS was used for the structural identification of R207910 and its metabolites. The (79)Br/(81)Br isotope ratio is also valuable in the search for metabolites in the complex background of endogenous compounds obtained using HPLC-ESI-MS analyses. Data-dependent scanning using isotope recognition with an ion trap mass spectrometer or processing of Q-Tof data provides HPLC-ICP-MS-like "bromatograms". The combination of accurate mass measurements and the fragmentation behavior in the MS(2) spectra obtained using the Q-Tof Ultima mass spectrometer or MS(n) spectra acquired using the LTQ-Orbitrap allowed structural characterization of the main metabolites of R207910 in methanolic dog and rat faeces extracts taken 0-24 h post-dose.

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

    ERIC Educational Resources Information Center

    Beussman, Douglas J.

    2007-01-01

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

  14. An Experiment Using Sucrose Density Gradients in the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Turchi, Sandra L.; Weiss, Monica

    1988-01-01

    Describes an experiment to be performed in an undergraduate biochemistry laboratory that is based on a gradient centrifugation system employing a simple bench top centrifuge, a freezer, and frozen surcose gradient solution to separate macromolecules and subcellular components. (CW)

  15. Synthesis and characterization of a multimode stationary phase: Congo red derivatized silica in nano-flow HPLC.

    PubMed

    Zhang, Yi; Zhang, Yan; Wang, Guan; Chen, Wujuan; He, Pingang; Wang, Qingjiang

    2016-02-01

    A novel Congo red (CR) derivatized silica stationary phase was prepared and packed into a fused silica capillary tube for nano-flow HPLC. A variety of analytes including poly-aromatic hydrocarbons, parabens, acids, sulfonamides, bases, and nucleosides were successfully separated using the CR. In comparison with commercial ODS columns, this new stationary phase has a different separation mechanism (hydrophobically-assisted ion-exchange), which was evident in the separation of benzoic acid derivatives and sulfonamides. The successful application of CR-bonded silica stationary phase in the HILIC and PALC modes demonstrates the effectiveness of this potential chromatographic material in nano flow HPLC.

  16. Synthesis and characterization of a multimode stationary phase: Congo red derivatized silica in nano-flow HPLC.

    PubMed

    Zhang, Yi; Zhang, Yan; Wang, Guan; Chen, Wujuan; He, Pingang; Wang, Qingjiang

    2016-02-01

    A novel Congo red (CR) derivatized silica stationary phase was prepared and packed into a fused silica capillary tube for nano-flow HPLC. A variety of analytes including poly-aromatic hydrocarbons, parabens, acids, sulfonamides, bases, and nucleosides were successfully separated using the CR. In comparison with commercial ODS columns, this new stationary phase has a different separation mechanism (hydrophobically-assisted ion-exchange), which was evident in the separation of benzoic acid derivatives and sulfonamides. The successful application of CR-bonded silica stationary phase in the HILIC and PALC modes demonstrates the effectiveness of this potential chromatographic material in nano flow HPLC. PMID:26646316

  17. Ultracapacitor separator

    DOEpatents

    Wei, Chang; Jerabek, Elihu Calvin; LeBlanc, Jr., Oliver Harris

    2001-03-06

    An ultracapacitor includes two solid, nonporous current collectors, two porous electrodes separating the collectors, a porous separator between the electrodes and an electrolyte occupying the pores in the electrodes and separator. The electrolyte is a polar aprotic organic solvent and a salt. The porous separator comprises a wet laid cellulosic material.

  18. HPLC Analysis of Chlorophyll a, Chlorophyll b, and Beta-Carotene in Collard Greens: A Project for a Problem-Oriented Laboratory Course.

    ERIC Educational Resources Information Center

    Silveira, Augustine, Jr.; And Others

    1984-01-01

    High performance liquid chromatography (HPLC) is used to separate and quantitate beta-carotene, chlorophyll a, and chlorophyll b originating from collard greens. Experimental procedures used and typical results obtained are discussed. (JN)

  19. Laser textured surface gradients

    NASA Astrophysics Data System (ADS)

    Ta, Van Duong; Dunn, Andrew; Wasley, Thomas J.; Li, Ji; Kay, Robert W.; Stringer, Jonathan; Smith, Patrick J.; Esenturk, Emre; Connaughton, Colm; Shephard, Jonathan D.

    2016-05-01

    This work demonstrates a novel technique for fabricating surfaces with roughness and wettability gradients and their subsequent applications for chemical sensors. Surface roughness gradients on brass sheets are obtained directly by nanosecond laser texturing. When these structured surfaces are exposed to air, their wettability decreases with time (up to 20 days) achieving both spatial and temporal wettability gradients. The surfaces are responsive to organic solvents. Contact angles of a series of dilute isopropanol solutions decay exponentially with concentration. In particular, a fall of 132° in contact angle is observed on a surface gradient, one order of magnitude higher than the 14° observed for the unprocessed surface, when the isopropanol concentration increased from 0 to 15.6 wt%. As the wettability changes gradually over the surface, contact angle also changes correspondingly. This effect offers multi-sensitivity at different zones on the surface and is useful for accurate measurement of chemical concentration.

  20. Development and validation of a rapid HPLC method for the quantification of GSE4 peptide in biodegradable PEI-PLGA nanoparticles.

    PubMed

    Egusquiaguirre, Susana P; Manguán-García, Cristina; Perona, Rosario; Pedraz, José Luís; Hernández, Rosa Maria; Igartua, Manuela

    2014-12-01

    In this work a high performance liquid chromatographic (HPLC) method has been developed and validated for the content determination of GSE4 peptide in PEI-PLGA nanoparticles. Chromatographic separation was performed on a C18 column, and a gradient elution with a mobile phase composed of methanol and 0.1% aqueous trifluoroacetic acid (TFA) solution, at a flow rate of 1ml/min, was used. GSE4 peptide identification was made by fluorescence detection at 290nm. The elution of methanol:TFA was initially maintained at (20:80, v/v) for one min and the gradient changed to (80:20, v/v) in 6min. This ratio was then followed by isocratic elution at (80:20, v/v) during another min and for further 3min it was linearly modified to (20:80, v/v). The developed method was validated according to the ICH guidelines, being specific, linear in the range 10-100μg/ml (R(2)=0.9996), precise, exhibiting good inter-day and intra-day precision reflected by the relative standard deviation values (less than 3.88%), accurate, with a recovery rate of 100.18±0.95%, and stable for 48h at 5°C or at RT when encapsulated in nanoparticles. The method was simple, fast, and successfully used to determine the peptide content in GSE4-loaded PEI-PLGA nanoparticles.

  1. Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS.

    PubMed

    Montoro, P; Carbone, V; Quiroz, J de Dioz Zuniga; De Simone, F; Pizza, C

    2004-01-01

    The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as 'uña de gato', have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC-ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on flow injection ES/MS, and a more complete analytical technique using HPLC-MS.

  2. Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS.

    PubMed

    Montoro, P; Carbone, V; Quiroz, J de Dioz Zuniga; De Simone, F; Pizza, C

    2004-01-01

    The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as 'uña de gato', have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC-ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on flow injection ES/MS, and a more complete analytical technique using HPLC-MS. PMID:14979528

  3. A Validated RP HPLC-PAD Method for the Determination of Hederacoside C in Ivy-Thyme Cough Syrup.

    PubMed

    Khdair, Ayman; Mohammad, Mohammad K; Tawaha, Khaled; Al-Hamarsheh, Eman; Alkhatib, Hatim S; Al-Khalidi, Bashar; Bustanji, Yasser; Najjar, Samer; Hudaib, Mohammad

    2010-01-01

    A simple reversed phase high-performance liquid chromatographic (RP-HPLC) method coupled with a photodiode array detector (PAD) has been developed and validated for the analysis of hederacoside C, the marker of ivy plant, in Ivy-Thyme cough syrup. Separation of hederacoside C was achieved using a Phenomenex-Gemini C18 column isothermally at 40°C. A mobile phase system constituted of solvent A (water: acetonitrile: orthophosphoric acid (85%), 860 : 140 : 2 v/v) and solvent B (acetonitrile: orthophosphoric acid (85%), 998 : 2 v/v) was used, at gradient conditions, at a flow rate of 1.5 mL/min. Analysis was performed using UV-detection (205 nm). The method was linear over the range (0.03-0.15) mg/mL of hederacoside C (r = 0.9992). Repeatability and intermediate precision were acceptable (RSD <2%). Limits of detection (LOD) and quantitation (LOQ) were 0.011 and 0.032 mg/mL, respectively. Percentage recovery was found to lie between 99.69% and 100.90% (RSD <2%). The method was also proved to be specific (peak-purity coefficient = 0.996).

  4. A Validated RP HPLC-PAD Method for the Determination of Hederacoside C in Ivy-Thyme Cough Syrup.

    PubMed

    Khdair, Ayman; Mohammad, Mohammad K; Tawaha, Khaled; Al-Hamarsheh, Eman; Alkhatib, Hatim S; Al-Khalidi, Bashar; Bustanji, Yasser; Najjar, Samer; Hudaib, Mohammad

    2010-01-01

    A simple reversed phase high-performance liquid chromatographic (RP-HPLC) method coupled with a photodiode array detector (PAD) has been developed and validated for the analysis of hederacoside C, the marker of ivy plant, in Ivy-Thyme cough syrup. Separation of hederacoside C was achieved using a Phenomenex-Gemini C18 column isothermally at 40°C. A mobile phase system constituted of solvent A (water: acetonitrile: orthophosphoric acid (85%), 860 : 140 : 2 v/v) and solvent B (acetonitrile: orthophosphoric acid (85%), 998 : 2 v/v) was used, at gradient conditions, at a flow rate of 1.5 mL/min. Analysis was performed using UV-detection (205 nm). The method was linear over the range (0.03-0.15) mg/mL of hederacoside C (r = 0.9992). Repeatability and intermediate precision were acceptable (RSD <2%). Limits of detection (LOD) and quantitation (LOQ) were 0.011 and 0.032 mg/mL, respectively. Percentage recovery was found to lie between 99.69% and 100.90% (RSD <2%). The method was also proved to be specific (peak-purity coefficient = 0.996). PMID:20862201

  5. [Simultaneous determination of 4 alkaloids and a flavonoid in Picrasmae Ramulus et Folium by RP-HPLC].

    PubMed

    Wu, Chong; Deng, Gui-Hua; Lin, Chao-Zhan; Zhu, Chen-Chen

    2014-05-01

    A RP-HPLC method was developed to evaluate the quality of Picrasmae Ramulus et Folium by simultaneous determination of five constituents including 1-hydroxymethyl-beta-carboline (1), 1-methoxicabony-beta-carboline (2), 4-methoxy-5-hydroxy-canthin-6-one (3), 4, 5-dimethoxy-canthin-6-one (4) and maackiain (5) in Picrasmae Ramulus et Folium. The samples were separated on a Kromasil RP-C18 (4.6 mm x 250 mm, 5 microm) column eluted with acetonitrile and 0.1% phosphoric acid as mobile phases in gradient mode. The detection wavelength was set at 254 nm. The calibration curves and linearity of the above five standards were determined as (1) Y = 6 525.6X + 37.25 (0.009-1.780 microg, r = 0.996 8), (2) Y = 3 662.3X + 41.55 (0.005-0.920 microg, r = 0.999 5), (3) Y = 3763.1X + 146.87 (0.015-3.060 microg, r = 0.999 0), (4) Y = 2 174.1X + 21.52 (0.003-0.620 microg, r = 0.999 5), and (5) Y = 276.25X + 7.65 (0.010-1.960 microg, r = 0.998 9), respectively. The method is simple and repeatable, and can be used for the quality assessment of Picrasmae Ramulus et Folium.

  6. Simultaneous determination of bioactive compounds in Piper nigrum L. and a species comparison study using HPLC-PDA.

    PubMed

    Rao, Vidadala Rama Subba; Raju, Sagi Satyanarayana; Sarma, Vanka Umamaheswara; Sabine, Fouriner; Babu, Kothapalli Hari; Babu, Katragadda Suresh; Rao, Janaswamy Madhusudana

    2011-08-01

    Piper nigrum L. is a traditional medicine widely used in India for illnesses such as constipation, diarrhoea, earache, gangrene, heart disease, hernia, hoarseness, indigestion, insect bites, insomnia, joint pain, liver problems, lung disease, oral abscesses, sunburn, tooth decay and toothaches. In this study, six bioactive compounds, namely piperine (1), pellitorine (2), guineensine (3), pipnoohine (4), trichostachine (5) and piperonal (6) were quantified in different extracts of P. nigrum L. and compared with those of P. longum L. and P. chaba Hunter. To evaluate the quality of P. nigrum, a simple, accurate and precise HPLC-PDA method was developed for the simultaneous determination of the above-mentioned six compounds. The separation was achieved by Phenomenex Luna RP C(18) column (150 × 4.6 mm, 5 µm, Phenomenex Inc, CA, USA) with a binary gradient solvent system of water-acetonitrile, at a flow rate of 1.0 mL min(-1) and detected at 210, 232, 262 and 343 nm. All six calibration curves showed good linearity (R (2) > 0.9966). The method was reproducible with intra- and inter-day variations of less than 2% and 5%, respectively. The results demonstrated that this method is simple, reliable and suitable for the quality control of these plants.

  7. Stability-Indicating RP-HPLC Method for Simultaneous Estimation of Enrofloxacin and Its Degradation Products in Tablet Dosage Forms.

    PubMed

    Chakravarthy, V Ashok; Sailaja, B B V; Kumar, Avvaru Praveen

    2015-01-01

    The present work was the development of a simple, efficient, and reproducible stability-indicating reverse-phase high performance liquid chromatographic (RP-HPLC) method for simultaneous determination enrofloxacin (EFX) and its degradation products including ethylenediamine impurity, desfluoro impurity, ciprofloxacin impurity, chloro impurity, fluoroquinolonic acid impurity, and decarboxylated impurity in tablet dosage forms. The separation of EFX and its degradation products in tablets was carried out on Kromasil C-18 (250 × 4.6 mm, 5 μm) column using 0.1% (v/v) TEA in 10 mM KH2PO4 (pH 2.5) buffer and methanol by linear gradient program. Flow rate was 1.0 mL min(-1) with a column temperature of 35°C and detection wavelength was carried out at 278 nm and 254 nm. The forced degradation studies were performed on EFX tablets under acidic, basic, oxidation, thermal, humidity, and photolytic conditions. The degraded products were well resolved from the main active drug and also from known impurities within 65 minutes. The method was validated in terms of specificity, linearity, LOD, LOQ, accuracy, precision, and robustness as per ICH guidelines. The results obtained from the validation experiments prove that the developed method is a stability-indicating method and suitable for routine analysis. PMID:25705547

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

    PubMed Central

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

    2013-01-01

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

  9. Preparation of (+)-trans-isoalliin and its isomers by chemical synthesis and RP-HPLC resolution.

    PubMed

    Jayathilaka, Lasanthi; Gupta, Shalini; Huang, Jin-Sheng; Lee, Jenny; Lee, Bao-Shiang

    2014-09-01

    Naturally occurring (+)-trans-isoalliin, (R(C)R(S))-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H2O2 produces a mixture of (-)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (-)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC.

  10. Preparation of (+)-Trans-Isoalliin and Its Isomers by Chemical Synthesis and RP-HPLC Resolution

    PubMed Central

    Jayathilaka, Lasanthi; Gupta, Shalini; Huang, Jin-Sheng; Lee, Jenny; Lee, Bao-Shiang

    2014-01-01

    Naturally occurring (+)-trans-isoalliin, (RCRS)-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H2O2 produces a mixture of (−)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (−)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC. PMID:25187757

  11. Chemotactic separation of enzymes.

    PubMed

    Dey, Krishna Kanti; Das, Sambeeta; Poyton, Matthew F; Sengupta, Samudra; Butler, Peter J; Cremer, Paul S; Sen, Ayusman

    2014-12-23

    We demonstrate a procedure for the separation of enzymes based on their chemotactic response toward an imposed substrate concentration gradient. The separation is observed within a two-inlet, five-outlet microfluidic network, designed to allow mixtures of active (ones that catalyze substrate turnover) and inactive (ones that do not catalyze substrate turnover) enzymes, labeled with different fluorophores, to flow through one of the inlets. Substrate solution prepared in phosphate buffer was introduced through the other inlet of the device at the same flow rate. The steady-state concentration profiles of the enzymes were obtained at specific positions within the outlets of the microchannel using fluorescence microscopy. In the presence of a substrate concentration gradient, active enzyme molecules migrated preferentially toward the substrate channel. The excess migration of the active enzyme molecules was quantified in terms of an enrichment coefficient. Experiments were carried out with different pairs of enzymes. Coupling the physics of laminar flow of liquid and molecular diffusion, multiphysics simulations were carried out to estimate the extent of the chemotactic separation. Our results show that, with appropriate microfluidic arrangement, molecular chemotaxis leads to spontaneous separation of active enzyme molecules from their inactive counterparts of similar charge and size.

  12. Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing.

    PubMed

    Wilson, Rachael E; Groskreutz, Stephen R; Weber, Stephen G

    2016-05-17

    Capillary HPLC (cLC) with gradient elution is the separation method of choice for the fields of proteomics and metabolomics. This is due to the complementary nature of cLC flow rates and electrospray or nanospray ionization mass spectrometry (ESI-MS). The small column diameters result in good mass sensitivity. Good concentration sensitivity is also possible by injection of relatively large volumes of solution and relying on solvent-based solute focusing. However, if the injection volume is too large or solutes are poorly retained during injection, volume overload occurs which leads to altered peak shapes, decreased sensitivity, and lower peak capacity. Solutes that elute early even with the use of a solvent gradient are especially vulnerable to this problem. In this paper, we describe a simple, automated instrumental method, temperature-assisted on-column solute focusing (TASF), that is capable of focusing large volume injections of small molecules and peptides under gradient conditions. By injecting a large sample volume while cooling a short segment of the column inlet at subambient temperatures, solutes are concentrated into narrow bands at the head of the column. Rapidly raising the temperature of this segment of the column leads to separations with less peak broadening in comparison to solvent focusing alone. For large volume injections of both mixtures of small molecules and a bovine serum albumin tryptic digest, TASF improved the peak shape and resolution in chromatograms. TASF showed the most dramatic improvements with shallow gradients, which is particularly useful for biological applications. Results demonstrate the ability of TASF with gradient elution to improve the sensitivity, resolution, and peak capacity of volume overloaded samples beyond gradient compression alone. Additionally, we have developed and validated a double extrapolation method for predicting retention factors at extremes of temperature and mobile phase composition. Using this method

  13. Determination of zinc pyrithione in shampoos by HPLC and HPLC-MS/MS.

    PubMed

    Gu, Yu-Xiang; Wang, Qing-He; Zhou, Ze-Lin; Lv, Qing; Mai, Cheng-Hua

    2014-01-01

    Methods have been developed for the determination of zinc pyrithione (ZPT) in shampoos using high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS). Samples were washed by water first to remove surfactant and water-soluble impurities, then ultrasonic-extracted by acetonitrile-methanol for 30 min, and finally analyzed by MG C18 column (250 mm x 4.6 mm, 5 μm) or RP-18e (100 mm x 3 mm, 2 μm) plus APCI-MS/MS. Limits of detection were determined as 0.015% (HPLC) and 0.003% (HPLC-MS/MS), with a limit of quantization of 0.05% and 0.01%, respectively. The recoveries were 85.8-104% (HPLC) and 87.6-107% (HPLC-MS/MS). A good linear relationship was obtained from 3.20 μg·ml(-1) to 200 μg·ml(-1) (HPLC) and 1.00 μg·ml(-1) to 200 μg·ml(-1) (HPLC-MS/MS). The proposed methods have been successfully applied to the analysis of ZPT in many shampoos. The established two methods were rapid and reproducible with low interference.

  14. Determination of zinc pyrithione in shampoos by HPLC and HPLC-MS/MS.

    PubMed

    Gu, Yu-Xiang; Wang, Qing-He; Zhou, Ze-Lin; Lv, Qing; Mai, Cheng-Hua

    2014-01-01

    Methods have been developed for the determination of zinc pyrithione (ZPT) in shampoos using high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS). Samples were washed by water first to remove surfactant and water-soluble impurities, then ultrasonic-extracted by acetonitrile-methanol for 30 min, and finally analyzed by MG C18 column (250 mm x 4.6 mm, 5 μm) or RP-18e (100 mm x 3 mm, 2 μm) plus APCI-MS/MS. Limits of detection were determined as 0.015% (HPLC) and 0.003% (HPLC-MS/MS), with a limit of quantization of 0.05% and 0.01%, respectively. The recoveries were 85.8-104% (HPLC) and 87.6-107% (HPLC-MS/MS). A good linear relationship was obtained from 3.20 μg·ml(-1) to 200 μg·ml(-1) (HPLC) and 1.00 μg·ml(-1) to 200 μg·ml(-1) (HPLC-MS/MS). The proposed methods have been successfully applied to the analysis of ZPT in many shampoos. The established two methods were rapid and reproducible with low interference. PMID:25682618

  15. Temperature gradients drive mechanical energy gradients in the flight muscle of Manduca sexta.

    PubMed

    George, N T; Sponberg, S; Daniel, T L

    2012-02-01

    A temperature gradient throughout the dominant flight muscle (dorsolongitudinal muscle, DLM(1)) of the hawkmoth Manduca sexta, together with temperature-dependent muscle contractile rates, demonstrates that significant spatial variation in power production is possible within a single muscle. Using in situ work-loop analyses under varying muscle temperatures and phases of activation, we show that regional differences in muscle temperature will induce a spatial gradient in the mechanical power output throughout the DLM(1). Indeed, we note that this power gradient spans from positive to negative values across the predicted temperature range. Warm ventral subunits produce positive power at their in vivo operating temperatures, and therefore act as motors. Concurrently, as muscle temperature decreases dorsally, the subunits produce approximately zero mechanical power output, acting as an elastic energy storage source, and negative power output, behaving as a damper. Adjusting the phase of activation further influences the temperature sensitivity of power output, significantly affecting the mechanical power output gradient that is expressed. Additionally, the separate subregions of the DLM(1) did not appear to employ significant physiological compensation for the temperature-induced differences in power output. Thus, although the components of a muscle are commonly thought to operate uniformly, a significant within-muscle temperature gradient has the potential to induce a mechanical power gradient, whereby subunits within a muscle operate with separate and distinct functional roles.

  16. An RP-HPLC determination of 5-hydroxymethylfurfural in honey The case of strawberry tree honey.

    PubMed

    Spano, Nadia; Casula, Lucia; Panzanelli, Angelo; Pilo, Maria I; Piu, Paola C; Scanu, Roberta; Tapparo, Andrea; Sanna, Gavino

    2006-02-15

    The use of the RP-HPLC official method of the International Honey Commission (IHC) for the determination of 5-hydroxymethylfurfural (HMF) in strawberry tree honey (Arbutus unedo, a typical Sardinian honey) has brought to light a specific and heavy chromatographic interference that prevents accurate quantification. The interference has been identified as homogentisic acid (HA), i.e. the marker of the botanical origin of the honey. For this reason, an alternative RP-HPLC method is proposed. The bias-free method allows a complete separation of HMF from HA to the baseline level and is faster and more precise than the RP-HPLC official method: the detection and quantification limits are 1.9 and 4.0mgkg(-1), respectively, whereas the repeatability is ca. 2% in the HMF concentration range of 5-140mgkg(-1). PMID:18970477

  17. Crosswind Shear Gradient Affect on Wake Vortices

    NASA Technical Reports Server (NTRS)

    Proctor, Fred H.; Ahmad, Nashat N.

    2011-01-01

    Parametric simulations with a Large Eddy Simulation (LES) model are used to explore the influence of crosswind shear on aircraft wake vortices. Previous studies based on field measurements, laboratory experiments, as well as LES, have shown that the vertical gradient of crosswind shear, i.e. the second vertical derivative of the environmental crosswind, can influence wake vortex transport. The presence of nonlinear vertical shear of the crosswind velocity can reduce the descent rate, causing a wake vortex pair to tilt and change in its lateral separation. The LES parametric studies confirm that the vertical gradient of crosswind shear does influence vortex trajectories. The parametric results also show that vortex decay from the effects of shear are complex since the crosswind shear, along with the vertical gradient of crosswind shear, can affect whether the lateral separation between wake vortices is increased or decreased. If the separation is decreased, the vortex linking time is decreased, and a more rapid decay of wake vortex circulation occurs. If the separation is increased, the time to link is increased, and at least one of the vortices of the vortex pair may have a longer life time than in the case without shear. In some cases, the wake vortices may never link.

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

    PubMed

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

    2011-10-01

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

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

    PubMed

    Vongsak, Boonyadist; Sithisarn, Pongtip; Gritsanapan, Wandee

    2014-08-01

    Moringa oleifera Lam. has been used as a traditional medicine for the treatment of numerous diseases. A simultaneous high-performance liquid chromatography (HPLC) analysis was developed and validated for the determination of the contents of crypto-chlorogenic acid, isoquercetin and astragalin, the primary antioxidative compounds, in M. oleifera leaves. HPLC analysis was successfully conducted by using a Hypersil BDS C18 column, eluted with a gradient of methanol-1% acetic acid with a flow rate of 1 mL/min, and detected at 334 nm. Parameters for the validation included linearity, precision, accuracy and limits of detection and quantitation. The developed HPLC method was precise, with relative standard deviation < 2%. The recovery values of crypto-chlorogenic acid, isoquercetin and astragalin in M. oleifera leaf extracts were 98.50, 98.47 and 98.59%, respectively. The average contents of these compounds in the dried ethanolic extracts of the leaves of M. oleifera collected from different regions of Thailand were 0.081, 0.120 and 0.153% (w/w), respectively. The developed HPLC method was appropriate and practical for the simultaneous analysis of crypto-chlorogenic acid, isoquercetin and astragalin in the leaf extract of M. oleifera. This work is valuable as guidance for the standardization of the leaf extracts and pharmaceutical products of M. oleifera. PMID:23828911

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

    PubMed

    Vongsak, Boonyadist; Sithisarn, Pongtip; Gritsanapan, Wandee

    2014-08-01

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

  1. Ceramic transactions: Functionally gradient materials. Volume 34

    SciTech Connect

    Holt, J.B.; Koizumi, Mitsue; Hirai, Toshio; Munir, Z.A.

    1993-01-01

    A functionally gradient material (FGM) is a composite that smoothly transitions from one material at one surface to another material at the opposite surface. Metals and ceramics are usually the materials that are combined in a controlled manner to optimize a specific property. The First International Symposium on Functionally Gradient Materials was held in Sendai, Japan, in August 1990. Contained in the present volume are the Proceedings of the Second International Symposium on Functionally Gradient Materials, presented at the Third International Ceramic Science and Technology Congress, held in San Francisco, CA, November 1-4, 1992. The papers presented here are divided into eight sections: the concept of FGM; mathematical modeling; methods of fabrication; material evaluation; applications; joining processes in FGM; process characterization; and design considerations. Separate abstracts are provided for each of the 54 papers.

  2. HIGH GRADIENT INDUCTION ACCELERATOR

    SciTech Connect

    Caporaso, G J; Sampayan, S; Chen, Y; Blackfield, D; Harris, J; Hawkins, S; Holmes, C; Krogh, M; Nelson, S; Nunnally, W; Paul, A; Poole, B; Rhodes, M; Sanders, D; Selenes, K; Sullivan, J; Wang, L; Watson, J

    2007-06-21

    A new type of compact induction accelerator is under development at the Lawrence Livermore National Laboratory that promises to increase the average accelerating gradient by at least an order of magnitude over that of existing induction machines. The machine is based on the use of high gradient vacuum insulators, advanced dielectric materials and switches and is stimulated by the desire for compact flash x-ray radiography sources. Research describing an extreme variant of this technology aimed at proton therapy for cancer will be described. Progress in applying this technology to several applications will be reviewed.

  3. Carbon-Isotopic Analysis of Individual Pigments by HPLC-Moving Wire-IRMS

    NASA Astrophysics Data System (ADS)

    Sessions, A. L.; Keely, B. J.; Hayes, J. M.

    2003-12-01

    We have developed a method for directly analyzing the carbon isotope ratios of individual pigments, including chlorophyll (chl) and its derivatives, by coupling a high-performance liquid chromatograph (HPLC) to an isotope-ratio mass spectrometer (IRMS) via a novel `moving-wire' interface. Pigments were separated on a reversed-phase C18 column, using a binary gradient modified from Airs et al. (2001, J. Chrom. A 917, 167-177). The HPLC effluent was dried onto a continuously-spooling nickel wire, and the involatile sample residue was combusted to CO2 and transferred to the IRMS for isotopic analysis. Replicate analyses of a standard solution yield precision for delta13C of better than 0.2‰ for injections containing ~5 μ g of chl-a. A five-fold improvement in sensitivity should be attainable using capillary HPLC to further reduce solvent volumes. The biomarker potential of tetrapyrrole pigments, combined with the geochemical information recorded by isotopic compositions, makes this combination a potent tool for biogeochemical studies. As a demonstration, we analyzed chlorophyll degradation products in sediments from a lake and a salina. First, compounds derived from bacteriochlorophylls (bchl)-c and -d were extracted from sediment cores taken at Kirisjes Lake (Larsmann Hills, Antarctica). These pigments are products of green sulfur bacteria and indicate the presence of an anoxic photic zone. The δ 13C values of bchl-related compounds are near -25‰ . Using published fractionations for Chlorobium species to extrapolate, dissolved CO2 in Kirisjes Lake probably had a δ 13C value of -12 to -21‰ and was strongly influenced by the recycling of organic carbon, possibly including methane. Second, compounds derived from chl-a, bchl-c, and bchl-d were isolated from sediments taken below a living microbial mat in the hypersaline les Salines de la Trinital (South Catalonia, Spain). The sediments contain visible remnants of past microbial mats and pigment distributions

  4. Determination of flavonoids and stilbenes in red wine and related biological products by HPLC and HPLC-ESI-MS-MS.

    PubMed

    Stecher, G; Huck, C W; Popp, M; Bonn, G K

    2001-09-01

    To investigate probable health benefits of flavonoids and stilbenes in red wine a new reversed-phase (RP) high-performance liquid-chromatographic (HPLC) method with enhanced separation efficiency and improved selectivity, sensitivity, and speed has been established for determination of the flavonoids quercetin, myricetin and kaempferol and the stilbenes cis- and trans-resveratrol, in a single run . UV-absorbance, fluorescence (FLD), and mass-spectrometric (MS) detection were also evaluated. UV-absorbance detection at 320 nm for stilbenes and 377 nm for flavonoids enables their determination up to the nanogram range with a linearity of R2>0.9999 (linear range 50 ng mL(-1)-50 microg mL(-1)). Calculated values of average recoveries were between 95 and 105% for all analytes. For resveratrol, fluorescence detection was highly selective and twice as sensitive as UV detection, and linearity was satisfactory (R2>0.9996; linear range see UV detection). For the detection of the hydrophilic glycosidic compounds piceid and rutin, which are coeluted with other hydrophilic ingredients, the validated RP HPLC system was coupled to a quadrupole ion-trap mass-spectrometer (MS) via an electrospray interface (ESI) with 25% ammonia solution as sheath liquid. MS detection was, highly linear (R2>0.9878; linear range 50 ng mL(-1)-50 microg mL(-1)) for all investigated analytes and the limits of detection were in the low nanogram range. Compared with UV detection MS detection resulted in a 200% increase in signal intensity for myricetin and 400% increases for quercetin and kaempferol, but equal signal intensity for resveratrol. Calculated values of average recoveries were 102% for myricetin and 79% for piceid. Collision induced dissociation (CID) was also used to obtain characteristic fragmentation fingerprints to facilitate qualitative and quantitative analysis even in complex matrices. Finally, this hyphenated HPLC-ESI-MS method was highly suitable and an essential improvement compared

  5. Validation a solid-phase extraction-HPLC method for determining the migration behaviour of five aromatic amines from packaging bags into seafood simulants.

    PubMed

    OuYang, Xiao-Kun; Luo, Yu-Yang; Wang, Yang-Guang; Yang, Li-Ye

    2014-01-01

    The simultaneous determination of five aromatic amines and their potential migration from packaging bags into seafood simulants were investigated. A validated HPLC method was developed for the separation and qualification of five aromatic amines in seafood simulants. By combining solid-phase extraction (SPE), these amines were efficiently separated on a Halo C18 column (150 × 4.6 mm i.d., 2.7 μm, particle size) using a mobile phase of methanol/phosphate buffer solution (5 mmol l(-1), pH 6.9) with gradient elution. The linear range was 0.1-10.0 mg l(-1); the absolute recoveries ranged from 85.3% to 98.4%; and the limits of detection of the five aromatic amines were between 0.015 and 0.08 mg l(-1). In this work the migration profile of aromatic amines from black plastic bags was investigated at temperatures of 4°C with water, 3% acetic acid solution, 10% ethanol solution and 50% ethanol solution as seafood simulants, respectively. The migration of the five aromatic amines under different conditions showed that residual o-methoxyaniline, p-chloroaniline, aniline and 2,6-dimethylaniline leaching from black plastic bags increased with incubation time. No detectable 3,3´-dimethylbenzidine was found to leach from the bags.

  6. Gradient Refractive Index Lenses.

    ERIC Educational Resources Information Center

    Morton, N.

    1984-01-01

    Describes the nature of gradient refractive index (GRIN) lenses, focusing on refraction in these materials, focal length of a thin Wood lens, and on manufacturing of such lenses. Indicates that GRIN lenses of small cross section are in limited production with applications suggested for optical communication and photocopying fields. (JN)

  7. HPLC determination of naproxen in plasma.

    PubMed

    Tashtoush, B M; Al-Taani, B M

    2003-09-01

    An assay method using isocratic HPLC with fluorometric detection for the determination of naproxen sodium in plasma is presented. A reverse phase Microbondapack column was used with a mobile phase consisting of 42% acetonitrile and 58% water adjusted to pH 3 using phosphoric acid. The fluorometric detector with an excitation wavelength of 270 nm and emission wavelength of 340 nm provided high sensitivity and no interferences from plasma constituents. Plasma samples were injected to HPLC without any extraction. The method was precise and reproducible as was demonstrated by replicate analysis of pooled plasma sample containing 0.5-80 microg/ml naproxen sodium.

  8. Arterial Stiffness Gradient

    PubMed Central

    Fortier, Catherine; Agharazii, Mohsen

    2016-01-01

    Background Aortic stiffness is a strong predictor of cardiovascular mortality in various clinical conditions. The aim of this review is to focus on the arterial stiffness gradient, to discuss the integrated role of medium-sized muscular conduit arteries in the regulation of pulsatile pressure and organ perfusion and to provide a rationale for integrating their mechanical properties into risk prediction. Summary The physiological arterial stiffness gradient results from a higher degree of vascular stiffness as the distance from the heart increases, creating multiple reflective sites and attenuating the pulsatile nature of the forward pressure wave along the arterial tree down to the microcirculation. The stiffness gradient hypothesis simultaneously explains its physiological beneficial effects from both cardiac and peripheral microcirculatory points of view. The loss or reversal of stiffness gradient leads to the transmission of a highly pulsatile pressure wave into the microcirculation. This suggests that a higher degree of stiffness of medium-sized conduit arteries may play a role in protecting the microcirculation from a highly pulsatile forward pressure wave. Using the ratio of carotid-femoral pulse wave velocity (PWV) to carotid-radial PWV, referred to as PWV ratio, a recent study in a dialysis cohort has shown that the PWV ratio is a better predictor of mortality than the classical carotid-femoral PWV. Key Messages Theoretically, the use of the PWV ratio seems more logical for risk determination than aortic stiffness as it provides a better estimation of the loss of stiffness gradient, which is the unifying hypothesis that explains the impact of aortic stiffness both on the myocardium and on peripheral organs. PMID:27195235

  9. A confirmatory HPLC-MS/MS method for ten synthetic corticosteroids in bovine urines.

    PubMed

    Savu, S R; Silvestro, L; Haag, A; Sörgel, F

    1996-12-01

    In the present study, an HPLC-MS/MS method to confirm, in bovine urine, the most common synthetic corticosteroids illegally used as growth promoters in livestock breeding will be presented. An API III-Plus (PE-Sciex) triple quadrupole mass spectrometer, interfaced by means of an atmospheric pressure chemical ionization source to the HPLC system, was used. Urine samples were treated with a sulfatase-glucuronidase mixture to cleave the drug-conjugates and then extracted on C18 disposable columns. LC separations were performed on a reversed-phase C18 column with ammonium acetate 0.1 M/acetonitrile (60/40, v/v) as mobile phase. Detection was performed in multiple reaction monitoring mode, negative ions, selecting fragmentations characteristic of 10 corticosteroids used more frequently. Good results, in terms of sensitivity and specificity have been obtained for nine corticosteroids that can be analyzed in the same HPLC run; the limits of sensitivity achieved were 0.05-1.0 ng/ml in urine. Only a more polar corticosteroid, required a different HPLC separation. Practical applications of this technique to real samples proved that it is an effective method to confirm the illegal use of corticosteroids as growth promoter in animal. In comparison with the chemical GC-MS methods the simpler sample preparation and the faster time of analysis permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity.

  10. Characterization and classification of stationary phases in HPLC and SFC – a review.

    PubMed

    Galea, Charlene; Mangelings, Debby; Vander Heyden, Yvan

    2015-07-30

    Packed column supercritical fluid chromatography (pSFC) is an attractive technique in drug discovery related analysis because it offers several advantages over the more commonly used high-performance liquid chromatography (HPLC) technique. The environmental-friendly CO2 mobile phase, the high-throughput capacity, the increased efficiency and the lower operational costs give SFC additional benefits over HPLC in analysis related to drug development. The latter technique is well established and has been used for decades in the pharmaceutical industry. On the other hand, SFC is still in its infancy, even though the technique has been known for decades and researchers are still discovering the possibilities and limitations of this technique. Column characterization aims to obtain a quantitative understanding of the properties of a column that influence the selectivity of a separation. Columns have been extensively characterized in HPLC using several approaches. However, limited column characterization has been done in SFC. Determining column properties allows the rapid selection of appropriate columns for method development for a particular application. Hence, they also show which columns will provide similar or dissimilar separations. The aim of this review is to compare and contrast approaches used to characterize stationary phases in HPLC and SFC, and to highlight points that still may need to be researched further. PMID:26320631

  11. LIQUID CHROMATOGRAPHIC SEPARATION OF THE ENANTIOMERS OF TRANS-CHLORDANE, CIS-CHLORDANE, HEPTACHLOR, HEPTACHLOR EPOXIDE AND ALPHA-HEXACHLOROCYCLOHEXANE WITH APPLICATION TO SMALL-SCALE PREPARATIVE SEPARATION

    EPA Science Inventory

    Analytical high-performance liquid chromatographic separations of the individual enantiomers of five polychlorinated compounds were obtained on polysaccharide stereoselective HPLC columns. The enantiomers of the pesticides trans-chlordane, cis-chlordane and heptachlor were separa...

  12. [Determination of 7 bio-active alkaloids in Stephania plants by RP-HPLC].

    PubMed

    Huang, J; Guo, J; Duan, G

    1998-07-01

    Seven bio-active alkaloids (stepholidine, sinoacutine, isocorydine, l-tetrahydropalmatine, crebanine, fanchinoline and tetrandrine) in Stephania plants were determined by RP-HPLC, using UV detection (282 nm) and gradient elution. The reversed phase system consisted of ODS column and methanol-water-triethylamine as mobile phase. The flow rate was 1.0 ml.min-1. Good linearity between peak heights and concentrations of the alkaloids was obtained in the concentration range. The HPLC method proved accurate, precise and sensitive. The results showed that there were some differences in the occurrence and content of the alkaloids between various species and between the same species from different habitats and collecting time. Based on the results, some species with high content of the 7 bio-active alkaloids were selected. The study provided some useful information for the utilization of medicinal plant resources in the genus Stephania. PMID:12016887

  13. Quantification of phytochelatins in plants by reversed-phase HPLC-ESI-MS-MS.

    PubMed

    El-Zohri, M H A; Cabala, R; Frank, H

    2005-08-01

    An on-line HPLC-ESI-MS-MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS-MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 micromol kg(-1) plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions. PMID:16001238

  14. Quantification of phytochelatins in plants by reversed-phase HPLC-ESI-MS-MS.

    PubMed

    El-Zohri, M H A; Cabala, R; Frank, H

    2005-08-01

    An on-line HPLC-ESI-MS-MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS-MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 micromol kg(-1) plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions.

  15. Coral Pigments: Quantification Using HPLC and Detection by Remote Sensing

    NASA Technical Reports Server (NTRS)

    Cottone, Mary C.

    1995-01-01

    Widespread coral bleaching (loss of pigments of symbiotic dinoflagellates), and the corresponding decline in coral reef health worldwide, mandates the monitoring of coral pigmentation. Samples of the corals Porites compressa and P. lobata were collected from a healthy reef at Puako, Hawaii, and chlorophyll (chl) a, peridinin, and Beta-carotene (Beta-car) were quantified using reverse-phase high performance liquid chromatography (HPLC). Detailed procedures are presented for the extraction of the coral pigments in 90% acetone, and the separation, identification, and quantification of the major zooxanthellar pigments using spectrophotometry and a modification of the HPLC system described by Mantoura and Llewellyn (1983). Beta-apo-8-carotenal was found to be inadequate as in internal standard, due to coelution with chl b and/or chl a allomer in the sample extracts. Improvements are suggested, which may result in better resolution of the major pigments and greater accuracy in quantification. Average concentrations of peridinin, chl a, and Beta-car in corals on the reef were 5.01, 8.59, and 0.29, micro-grams/cm(exp 2), respectively. Average concentrations of peridinin and Beta-car did not differ significantly between the two coral species sampled; however, the mean chl a concentration in P. compressa specimens (7.81 ,micro-grams/cm(exp 2) was significantly lower than that in P. lobata specimens (9.96 11g/cm2). Chl a concentrations determined spectrophotometrically were significantly higher than those generated through HPLC, suggesting that spectrophotometry overestimates chl a concentrations. The average ratio of chl a-to-peridinin concentrations was 1.90, with a large (53%) coefficient of variation and a significant difference between the two species sampled. Additional data are needed before conclusions can be drawn regarding average pigment concentrations in healthy corals and the consistency of the chl a/peridinin ratio. The HPLC pigment concentration values

  16. A highly sensitive HPLC-MS/MS method for quantification of 20(S)-protopanaxadiol in human plasma and its application in phase IIa clinical trial of a novel antidepressant agent.

    PubMed

    Ling, Jin; Yu, Yingjia; Zhu, Jiajun; Li, Yan; Ling, Li; Wang, Liping; Xu, Changjiang; Duan, Gengli

    2016-09-15

    A highly sensitive HPLC-MS/MS assay method was established to quantify 20(S)-protopanaxadiol (PPD) in human plasma with dexamethasone as an internal standard. The electrospray ion mass spectrometry (ESI-MS) was operated under the multiple reactions monitoring mode (MRM) using positive ion mode. PPD was extracted from 500μL plasma samples by liquid-liquid extraction then separated by a C18 analytical column with gradient elution. The concentration of PPD could be determined by this HPLC-MS/MS method over the range of 0.05-20ng/mL with the lower limit of quantification (LLOQ) of 0.05ng/mL. The method was successfully applied to phase IIa clinical trial of Yuxintine (PPD capsule) in which plasma samples of 87 subjects were analyzed following 6 weeks of oral administration of placebo or PPD capsules in 5 different doses. In this study, the measured concentration was linearly related to the oral dosage with R=0.9901. The minimum and maximum values of measured concentration were 0.06 and 11.60ng/mL, respectively. In addition, plasma concentrations of PPD in depression patients were reported for the first time in our study. PMID:27507666

  17. Simultaneous determination of five free and total flavonoids in rat plasma by ultra HPLC-MS/MS and its application to a comparative pharmacokinetic study in normal and hyperlipidemic rats.

    PubMed

    Wang, Xiaofan; Zhao, Xu; Gu, Liqiang; Lv, Chunxiao; He, Bosai; Liu, Zhenzhen; Hou, Pengyi; Bi, Kaishun; Chen, Xiaohui

    2014-03-15

    A simple and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) method has been developed for the simultaneous determination of five free flavonoids (amentoflavone, isorhamnetin, naringenin, kaempferol and quercetin) and their total (free and conjugated) forms, and to compare the pharmacokinetics of these active ingredients in normal and hyperlipidemic rats. The free and total forms of these flavonoids were extracted by liquid-liquid extraction with ethyl acetate. The conjugated flavonoids were deconjugated by the enzyme β-Glucuronidase and Sulfatase. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-C8 USP L7 column using gradient elution. Detection was performed on a 4000Q uHPLC-MS/MS system from AB Sciex using negative ion mode in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were 2.0-5.0ng/mL for all the analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from -9.3% to 11.0%, and the mean extraction recoveries of analytes and internal standard (IS) from rat plasma were all more than 81.7%. The validated method was successfully applied to a comparative pharmacokinetic study of five free and total analytes in rat plasma. The results indicated that the absorption of five total flavonoids in hyperlipidemia group were significantly higher than those in normal group with similar concentration-time curves. PMID:24566333

  18. Validated HPLC method for quantifying permethrin in pharmaceutical formulations.

    PubMed

    García, E; García, A; Barbas, C

    2001-03-01

    An isocratic HPLC method for permethrin determination in raw material and pharmaceutical presentations as lotion and shampoo has been developed and validated following ICH recommendations. Cis and trans- isomers, impurities and degradation products are well separated. The chromatographic analysis were performed on a 4 microm particle C-18 Nova-Pak (Waters, Madrid, Spain) column (15 x 0.39 cm) kept in a Biorad column oven at 35 degrees C. Mobile phase consisted of methanol--water (78:22, v/v) at a flow rate of 1 ml/min. UV detection was performed at 272 nm and peaks were identified with retention times as compared with standards and confirmed with characteristic spectra using the photodiode array detector.

  19. Characterization of nutraceuticals and functional foods by innovative HPLC methods.

    PubMed

    Corradini, Claudio; Galanti, Roberta; Nicoletti, Isabella

    2002-04-01

    In recent years there is a growing interest in food and food ingredient which may provide health benefits. Food as well as food ingredients containing health-preserving components, are not considered conventional food, but can be defined as functional food. To characterise such foods, as well as nutraceuticals specific, high sensitive and reproducible analytical methodologies are needed. In light of this importance we set out to develop innovative HPLC methods employing reversed phase narrow bore column and high-performance anion-exchange chromatographic methods coupled with pulsed amperometric detection (HPAEC-PAD), which are specific for carbohydrate analysis. The developed methods were applied for the separation and quantification of citrus flavonoids and to characterize fructooligosaccharide (FOS) and fructans added to functional foods and nutraceuticals.

  20. Contamination in HPLC quantified benzaldehyde is from polypropylene microtubes.

    PubMed

    Shemesh, Tomer; Karschimkus, Connie; Rowley, Kevin G

    2005-11-01

    Semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6) is a copper-containing enzyme predominantly expressed by vascular smooth muscle cells. SSAO deaminates primary amines to produce aldehydes and oxygen peroxides, and may thus play a role in vascular damage. SSAO activity can be quantified by assaying benzaldehyde production using fluorescent derivatisation and separation by HPLC. We performed the derivatisation step in polypropylene or borosilicate glass tubes over 45 min at 95 degrees C. High and obstructing background levels of benzaldehyde were found in one batch of polypropylene vials, as opposed to its alternatives. Treatment and handling of product shipment into the country did not account for introduction of contaminant into packaged vials nor did any reagent used in the assay. We conclude that the source of contamination was most likely due to variation in the commercial production process. Use of borosilicate vials for assays based on aldehyde production and derivatisation is recommended.

  1. Stress-gradient plasticity

    PubMed Central

    Chakravarthy, Srinath S.; Curtin, W. A.

    2011-01-01

    A new model, stress-gradient plasticity, is presented that provides unique mechanistic insight into size-dependent phenomena in plasticity. This dislocation-based model predicts strengthening of materials when a gradient in stress acts over dislocation source–obstacle configurations. The model has a physical length scale, the spacing of dislocation obstacles, and is validated by several levels of discrete-dislocation simulations. When incorporated into a continuum viscoplastic model, predictions for bending and torsion in polycrystalline metals show excellent agreement with experiments in the initial strengthening and subsequent hardening as a function of both sample-size dependence and grain size, when the operative obstacle spacing is proportional to the grain size. PMID:21911403

  2. Hand-Portable Gradient Capillary Liquid Chromatography Pumping System.

    PubMed

    Sharma, Sonika; Plistil, Alex; Barnett, Hal E; Tolley, H Dennis; Farnsworth, Paul B; Stearns, Stanley D; Lee, Milton L

    2015-10-20

    In this work, a novel splitless nanoflow gradient generator integrated with a stop-flow injector was developed and evaluated using an on-column UV-absorption detector. The gradient pumping system consisted of two nanoflow pumps controlled by micro stepper motors, a mixer connected to a serpentine tube, and a high-pressure valve. The gradient system weighed only 4 kg (9 lbs) and could generate up to 55 MPa (8000 psi) pressure. The system could operate using a 24 V DC battery and required 1.2 A for operation. The total volume capacity of the pump was 74 μL, and a sample volume of 60 nL could be injected. The system provided accurate nanoflow rates as low as 10 nL/min without employing a splitter, making it ideal for capillary column use. The gradient dwell volume was calculated to be 1.3 μL, which created a delay of approximately 4 min with a typical flow rate of 350 nL/min. Gradient performance was evaluated for gradient step accuracy, and excellent reproducibility was obtained in day-to-day experiments (RSD < 1.2%, n = 4). Linear gradient reproducibility was tested by separating a three-component pesticide mixture on a poly(ethylene glycol) diacrylate (PEGDA) monolithic column. The retention time reproducibility was very good in run-to-run experiments (RSD < 1.42%, n = 4). Finally, excellent separation of five phenols was demonstrated using the nanoflow gradient system.

  3. Identification and determination of major constituents in a traditional Chinese medicine compound recipe Xiongdankaiming tablet using HPLC-PDA/ESI-MSn and HPLC-UV/ELSD* #

    PubMed Central

    Wang, Shu-fang; Leng, Jing; Xu, Yi-min; Feng, Mei-ling

    2013-01-01

    Xiongdankaiming tablet (XDKMT), a well-known compound in traditional Chinese medicine, is widely used for the treatment of acute iridocyclitis and primary open-angle glaucoma. In this paper, accurate and reliable methods were developed for the identification of 20 constituents using high-performance liquid chromatography with photo-diode array and electron spray ionization-mass spectrometry (HPLC-PDA/ESI-MSn), and determination of nine of the constituents (chlorogenic acid, gentiopicroside, isochlorogenic acid B, diosmetin-7-O-β-D-glucopyranoside, apigenin, diosmetin, tauroursodeoxycholic acid, acacetin, and taurochenodeoxycholic acid) was developed using HPLC with ultraviolet absorption detector and evaporative light scattering detector (HPLC-UV/ELSD) for the first time. The best results were obtained on a Zorbax SB-C18 column with gradient elution using water (0.1% formic acid) (A) and methanol (0.1% formic acid) (B) at a flow rate of 0.7 ml/min. Tauroursodeoxycholic acid and taurochenodeoxycholic acid, owing to their low UV absorption, were detected by ELSD. The other seven compounds were analyzed by HPLC-UV with variable wavelengths. The calibration curves of all nine constituents showed good linear regression (R 2>0.9996) within the linearity ranges. The limits of detection and quantification were in the ranges of 0.0460–9.90 μg/ml and 0.115–24.8 μg/ml, respectively. The accuracy, in terms of recovery, varied from 95.3% to 104.9% with relative standard deviations (RSDs) less than 4.4%. Precision (with the intra- and inter-day variations less than 4.4%) was also suitable for its intended use. The developed method was successfully applied for the analysis of major components in XDKMT, which provides an appropriate method for the quality control of XDKMT. PMID:23825146

  4. Identification and determination of major constituents in a traditional Chinese medicine compound recipe Xiongdankaiming tablet using HPLC-PDA/ESI-MS(n) and HPLC-UV/ELSD.

    PubMed

    Wang, Shu-Fang; Leng, Jing; Xu, Yi-Min; Feng, Mei-Ling

    2013-07-01

    Xiongdankaiming tablet (XDKMT), a well-known compound in traditional Chinese medicine, is widely used for the treatment of acute iridocyclitis and primary open-angle glaucoma. In this paper, accurate and reliable methods were developed for the identification of 20 constituents using high-performance liquid chromatography with photo-diode array and electron spray ionization-mass spectrometry (HPLC-PDA/ESI-MS(n)), and determination of nine of the constituents (chlorogenic acid, gentiopicroside, isochlorogenic acid B, diosmetin-7-O-β-d-glucopyranoside, apigenin, diosmetin, tauroursodeoxycholic acid, acacetin, and taurochenodeoxycholic acid) was developed using HPLC with ultraviolet absorption detector and evaporative light scattering detector (HPLC-UV/ELSD) for the first time. The best results were obtained on a Zorbax SB-C18 column with gradient elution using water (0.1% formic acid) (A) and methanol (0.1% formic acid) (B) at a flow rate of 0.7 ml/min. Tauroursodeoxycholic acid and taurochenodeoxycholic acid, owing to their low UV absorption, were detected by ELSD. The other seven compounds were analyzed by HPLC-UV with variable wavelengths. The calibration curves of all nine constituents showed good linear regression (R(2)>0.9996) within the linearity ranges. The limits of detection and quantification were in the ranges of 0.0460-9.90 μg/ml and 0.115-24.8 μg/ml, respectively. The accuracy, in terms of recovery, varied from 95.3% to 104.9% with relative standard deviations (RSDs) less than 4.4%. Precision (with the intra- and inter-day variations less than 4.4%) was also suitable for its intended use. The developed method was successfully applied for the analysis of major components in XDKMT, which provides an appropriate method for the quality control of XDKMT.

  5. [ILs-HPLC simultanesous determination of five alkaloids in phellodenddri chinensis cortex].

    PubMed

    Jiang, Xin-Yi; Zhang, Hui-Fen; Wang, Sheng-Nan; Chen, Xiao-Hui

    2014-10-01

    A RP-HPLC method was established for simultaneous determination of phellodendrine hydrochloride (PH1), magnoflorine hydrochloride (MH), jatrorrhizine hydrochloride (JH), palmatine hydrochloride (PH2) and berberine hydrochloride (BH) in Phellodendri Chinensis Cortex by using ionic liquids as mobile phase additives. The separation was performed on a Kromasil C18 (4.6 mm x 250 mm, 5 μm) coupled with ultraviolet (UV) detection. The effect of extraction solvent, detection wavelength, length of alkyl chain on different imidazolium ionic liquids and concentration of ionic liquids on the separation and determination of alkaloids were investigated. Ionic liquid, [BMIm] BF4, can obviously improve the resolution and peak shape. This ILs-HPLC method is simple, rapid, and reliable, which can be used for determination of alkaloids in Phellodenddri Chinensis Cortex. PMID:25612445

  6. Monitoring of aminophenol isomers in surface water samples using a new HPLC method.

    PubMed

    Badea, Irinel Adriana; Axinte, Lacramioara; Vladescu, Luminita

    2013-03-01

    A new HPLC method was developed for the simultaneous determination of aminophenol isomers by means of a mixed-mode stationary phase containing both SCX and C18 moieties. All factors influencing the separation were discussed and optimized. The chromatographic conditions for the separation of aminophenols are the stationary phase duet SCX/C18, the mobile phase of aqueous phosphate buffer (pH 4.85):methanol = 85:15 (v/v) delivered with a flow rate of 1 mL/min and a detection at 285 nm. The method proposed was validated in terms of linearity, limits of detection and quantification, accuracy and precision. The HPLC method elaborated here was applied with good results on river water samples. In order to survey the quality of surface rivers entered in treatment plants which deliver water for Bucharest, two major rivers were included in a monitoring program which last more than 1 year.

  7. Char separator

    DOEpatents

    Matthews, Francis T.

    1979-01-01

    Particulates removed from the flue gases produced in a fluidized-bed furnace are separated into high-and low-density portions. The low-density portion is predominantly char, and it is returned to the furnace or burned in a separate carbon burnup cell. The high-density portion, which is predominantly limestone products and ash, is discarded or reprocessed. According to another version, the material drained from the bed is separated, the resulting high-and low-density portions being treated in a manner similar to that in which the flue-gas particulates are treated.

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

    PubMed

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

    2015-09-11

    Porous metal-organic frameworks (MOFs) [Cu3(BTC)2(H2O)3]n (also known as HKUST-1; BTC, benzene-1,3,5-tricarboxylic acid) were synthesized as homogeneous shell onto carboxyl functionalized magnetic microparticles through a liquid phase epitaxy (LPE) process. The as-synthesized core-shell HKUST-1 magnetic microparticles composites were characterized by XRD and SEM, and used as stationary phase in high performance liquid chromatography (HPLC). The effects of the unique properties of MOFs onto the chromatographic performance are demonstrated by the experiments. First, remarkable separation of pyridine and bipyridine is achieved, although both molecules show a strong interaction between the Cu-ions in HKUST-1 and the nitrogen atoms in their heterocyles. The difference can be explained due to size exclusion of bipyridine from the well defined pore structure of crystalline HKUST-1. Second, the enormous variety of possible interactions of sample molecules with the metal ions and linkers within MOFs allows for specifically tailored solid phases for challenging separation tasks. For example, baseline separation of three chloroaniline (CLA) isomers tested can be achieved without the need for gradient elution modes. Along with the experimental HPLC runs, in-depth modelling with a recently developed chromatography modelling software (ChromX) was applied and proofs the software to be a powerful tool for exploring the separation potential of thin MOF films. The pore diffusivity of pyridine and CLA isomers within HKUST-1 are found to be around 2.3×10(-15)m(2)s(-1). While the affinity of HKUST-1 to the tested molecules strongly differs, the maximum capacities are in the same range, with 0.37molL(-1) for pyridine and 0.23molL(-1) for CLA isomers, corresponding to 4.0 and 2.5 molecules per MOF unit cell, respectively. PMID:26277028

  9. Validated HPLC and Ultra-HPLC Methods for Determination of Dronedarone and Amiodarone Application for Counterfeit Drug Analysis.

    PubMed

    El-Bagary, Ramzia I; Elkady, Ehab F; Mowaka, Shereen; Attallah, Maria

    2015-01-01

    Two simple, accurate, and precise chromatographic methods have been developed and validated for the determination of dronedarone (DRO) HCl and amiodarone (AMI) HCl either alone or in binary mixtures due to the possibility of using AMI as a counterfeit of DRO because of its lower price. First, an RP-HPLC method is described for the simultaneous determination of DRO and AMI. Chromatographic separation was achieved on a BDS Hypersil C18 column (150×4.6 mm, 5 μm). Isocratic elution based on potassium dihydrogen phosphate buffer with 0.1% triethylamine pH 6-methanol (10+90, v/v) at a flow rate of 2 mL/min with UV detection at 254 nm was performed. The second method is RP ultra-HPLC in which the chromatographic separation was achieved on an AcclaimTM RSLC 120 C18 column (100×2.1 mm, 2.2 μm) using isocratic elution with potassium dihydrogen phosphate buffer with 0.1% triethylamine pH 6-methanol (5+95, v/v) at a flow rate of 1 mL/min with UV detection at 254 nm. Linearity, accuracy, and precision of the two methods were found to be acceptable over the concentration ranges of 5-80 μg/mL for both DRO and AMI. The results were statistically compared using one-way analysis of variance. The optimized methods were validated and proved to be specific, robust, precise, and accurate for the QC of the drugs in their pharmaceutical preparations. PMID:26651561

  10. Efficient HPLC purification of endohedral metallofullerenes on a porphyrin-silica stationary phase

    SciTech Connect

    Xiao, Jie; Savina, M.R.; Martin, G.B.; Francis, A.H.; Meyerhoff, M.E. )

    1994-10-05

    Endohedral metallofullrenes are among the most fascinating materials to emerge from ongoing efforts to produce and characterize closed-cage carbon compounds. In this paper, we describe the use of a new tetraphenylporphyrin-silica support material for efficient HPLC purification of the metallofullerenes from crude soot extract. The true single-stage separation from pyridine extracts demonstrates the unique chemical selectivity exhibited by employing [pi]-electron-rich tetraphenylporphyrin-silica stationary phases in chemical purifications. 18 refs., 3 figs.

  11. Stereoisomers Separation

    NASA Astrophysics Data System (ADS)

    Wieczorek, Piotr

    The use of capillary electrophoresis for enantiomer separation and optical purity determination is presented. The contents start with basic information about the nature of stereoizomers and the mechanism of enantioseparation using capillary electrophoresis techniques. The molecules to be separated show identical chemical structure and electrochemical behavior. Therefore, the chiral recognition of enantiomers is possible only by bonding to chiral selector and the separation based on very small differences in complexation energies of diastereomer complexes formed. This method is useful for this purpose due to the fact that different compounds can be used as chiral selectors. The mostly used chiral selectors like cyclodextrins, crown ethers, chiral surfactants, macrocyclic antibiotics, transition metal complexes, natural, and synthetic polymers and their application for this purpose is also discussed. Finally, examples of practical applications of electromigration techniques for enantiomers separation and determination are presented.

  12. HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF THE ENANTIOMERS OF ORGANOPHOSPHORUS PESTICIDES ON POLYSACCHARIDE CHIRAL STATIONARY PHASES

    EPA Science Inventory

    High-performance liquid chromatographic separation of the individual enantiomers of 12 organophosphorus pesticides (OPs) was obtained on polysaccharide enantioselective HPLC columns using alkane-alcohol mobile phase. The OP pesticides were crotoxyphos, dialifor, fonofos, fenamiph...

  13. Product separator

    DOEpatents

    Welsh, Robert A.; Deurbrouck, Albert W.

    1976-01-20

    A secondary light sensitive photoelectric product separator for use with a primary product separator that concentrates a material so that it is visually distinguishable from adjacent materials. The concentrate separation is accomplished first by feeding the material onto a vibratory inclined surface with a liquid flow, such as a wet concentrating table. Vibrations generally perpendicular to the stream direction of flow cause the concentrate to separate from its mixture according to its color. When the concentrate and its surrounding stream reach the recovery end of the table, a detecting device notes the line of color demarcation and triggers a signal if it differs from a normal condition. If no difference is noted nothing moves on the second separator. However, if a difference is detected in the constant monitoring of the color line's location, a product splitter and recovery unit normally positioned near the color line at the recovery end, moves to a new position. In this manner the selected separated concentrate is recovered at a maximum rate regardless of variations in the flow stream or other conditions present.

  14. Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors.

    PubMed

    Campos, Patrícia Mazureki; Praça, Fabíola Silva Garcia; Bentley, Maria Vitória Lopes Badra

    2016-04-15

    Lipoic acid (LA) is an endogenous organosulfur compound with potent antioxidant property. LA is often used as a drug for the treatment of skin disorders. For the accomplishment of topical applications of LA appropriate drug quantification methods are essential. Thus far, no HPLC methods have been reported for the measurement of LA extracted from skin. In this article we report on the development and validation of three sensitive and specific HPLC methods for LA and dihydrolipoic acid (DHLA) using ultraviolet (UV), electrochemical (EC) or evaporative light scattering (ELS) detection. These methods demonstrate different linearity ranges. The chromatographic separations were performed by RP-HPLC (250 × 4 mm, 5 μm) with isocratic elution using an acidic mobile phase for the three detection techniques. The lower limits of detection and quantification were 0.04 and 0.08 ng LA, respectively, for HPLC coupled to ELS, an innovative detector for LA with high sensitivity. The extraction of LA from skin samples showed recoveries greater than 71%. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 μg/mL, respectively for HPLC/UV and 6.52 ± 0.49 and 5.01 ± 0.41 μg/mL, respectively, for HPLC/EC for the added LA concentration (6.67 μg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 μg/mL, respectively, for HPLC/ELS for the added LA concentration (10 μg/mL). These three optimized HPLC methods allowed for a simple, rapid and reliable determination of LA in human skin. They should be useful for the development of drug delivery systems for topical applications of LA.

  15. Gradient echo MRI

    PubMed Central

    Copenhaver, B R.; Shin, J; Warach, S; Butman, J A.; Saver, J L.; Kidwell, C S.

    2009-01-01

    Background: Recent studies have demonstrated that gradient echo (GRE) MRI sequences are as accurate as CT for the detection of intracerebral hemorrhage (ICH) in the context of acute stroke. However, many physicians who currently read acute stroke imaging studies may be unfamiliar with interpretation of GRE images. Methods: An NIH Web-based training program was developed including a pretest, tutorial, and posttest. Physicians involved in the care of acute stroke patients were encouraged to participate. The tutorial covered acute, chronic, and mimic hemorrhages as they appear on CT, diffusion-weighted imaging, and GRE sequences. Ability of users to identify ICH presence, type, and age on GRE was compared from the pretest to posttest timepoint. Results: A total of 104 users completed the tutorial. Specialties represented included general radiology (42%), general neurology (16%), neuroradiology (15%), stroke neurology (14%), emergency medicine (1%), and other (12%). Median overall score improved pretest to posttest from 66.7% to 83.3%, p < 0.001. Improvement by category was as follows: acute ICH, 66.7%–100%, p < 0.001; chronic ICH, 33.3%–66.7%, p < 0.001; ICH negatives/mimics, 100%–100%, p = 0.787. Sensitivity for identification of acute hemorrhage improved from 68.2% to 96.4%. Conclusions: Physicians involved in acute stroke care achieved significant improvement in gradient echo (GRE) hemorrhage interpretation after completing the NIH GRE MRI tutorial. This indicates that a Web-based tutorial may be a viable option for the widespread education of physicians to achieve an acceptable level of diagnostic accuracy at reading GRE MRI, thus enabling confident acute stroke treatment decisions. GLOSSARY AHA/ASA = American Heart Association/American Stroke Association; CME = continuing medical education; DWI = diffusion-weighted imaging; GRE = gradient echo; ICH = intracerebral hemorrhage; tPA = tissue plasminogen activator. PMID:19414724

  16. Gradient equivalent crystal theory.

    PubMed

    Zypman, F R; Ferrante, J

    2006-07-01

    This paper presents an extension of the formalism of equivalent crystal theory (ECT) by introducing an electron density gradient term so that the total model density becomes a more accurate representation of the real local density. Specifically, we allow for the electron density around a lattice site to have directionality, in addition to an average value, as assumed in ECT. We propose that an atom senses its neighbouring density as a weighted sum-the weights given by the its own electronic probability. As a benchmark, the method is used to compute vacancy migration energy curves of iron. These energies are in good agreement with previously published results. PMID:21690822

  17. Simultaneous determination of synthetic colorants in yogurt by HPLC.

    PubMed

    de Araújo Siqueira Bento, Waleska; Lima, Bruno Parente; Paim, Ana Paula S

    2015-09-15

    This article reports on a method to determine synthetic dyes in yogurts and milk drinks. Initially a method for extraction of artificial dyes was developed to pretreat samples in order to extract most of the artificial colorants. Then, the colorants were analyzed by HPLC-PAD using gradient elutions. The method was linear in the range of 0.5-25mgL(-1) colorants (0.9991

  18. Simultaneous determination of synthetic colorants in yogurt by HPLC.

    PubMed

    de Araújo Siqueira Bento, Waleska; Lima, Bruno Parente; Paim, Ana Paula S

    2015-09-15

    This article reports on a method to determine synthetic dyes in yogurts and milk drinks. Initially a method for extraction of artificial dyes was developed to pretreat samples in order to extract most of the artificial colorants. Then, the colorants were analyzed by HPLC-PAD using gradient elutions. The method was linear in the range of 0.5-25mgL(-1) colorants (0.9991

  19. Escalation of polymerization in a thermal gradient

    PubMed Central

    Mast, Christof B.; Schink, Severin; Gerland, Ulrich; Braun, Dieter

    2013-01-01

    For the emergence of early life, the formation of biopolymers such as RNA is essential. However, the addition of nucleotide monomers to existing oligonucleotides requires millimolar concentrations. Even in such optimistic settings, no polymerization of RNA longer than about 20 bases could be demonstrated. How then could self-replicating ribozymes appear, for which recent experiments suggest a minimal length of 200 nt? Here, we demonstrate a mechanism to bridge this gap: the escalated polymerization of nucleotides by a spatially confined thermal gradient. The gradient accumulates monomers by thermophoresis and convection while retaining longer polymers exponentially better. Polymerization and accumulation become mutually self-enhancing and result in a hyperexponential escalation of polymer length. We describe this escalation theoretically under the conservative assumption of reversible polymerization. Taking into account the separately measured thermophoretic properties of RNA, we extrapolate the results for primordial RNA polymerization inside a temperature gradient in pores or fissures of rocks. With a dilute, nanomolar concentration of monomers the model predicts that a pore length of 5 cm and a temperature difference of 10 K suffice to polymerize 200-mers of RNA in micromolar concentrations. The probability to generate these long RNAs is raised by a factor of >10600 compared with polymerization in a physical equilibrium. We experimentally validate the theory with the reversible polymerization of DNA blocks in a laser-driven thermal trap. The results confirm that a thermal gradient can significantly enlarge the available sequence space for the emergence of catalytically active polymers. PMID:23630280

  20. [Determination of azoxystrobin in tea by HPLC].

    PubMed

    Chonan, T

    2001-08-01

    A determination method has been developed for azoxystrobin in tea by HPLC. Azoxystrobin was extracted from a sample with acetone, and the extract was passed through an alumina column to remove tannin. The eluate was concentrated to ca. 25 mL and passed through a Sep-Pak Vac tC18 to remove pigments. The eluate was cleaned-up by using liquid-liquid partition, and Florisil and silica-gel columns. The HPLC analysis for azoxystrobin was carried out on a C18 column with acetonitrile-water (9:11) as the mobile phase, with ultraviolet detection at 260 nm. The recovery of azoxystrobin fortified at the level of 0.4 microgram/g was 90.2% and the limit of determination was 0.2 microgram/g.

  1. Map Separates

    USGS Publications Warehouse

    ,

    2001-01-01

    U.S. Geological Survey (USGS) topographic maps are printed using up to six colors (black, blue, green, red, brown, and purple). To prepare your own maps or artwork based on maps, you can order separate black-and-white film positives or negatives for any color printed on a USGS topographic map, or for one or more of the groups of related features printed in the same color on the map (such as drainage and drainage names from the blue plate.) In this document, examples are shown with appropriate ink color to illustrate the various separates. When purchased, separates are black-and-white film negatives or positives. After you receive a film separate or composite from the USGS, you can crop, enlarge or reduce, and edit to add or remove details to suit your special needs. For example, you can adapt the separates for making regional and local planning maps or for doing many kinds of studies or promotions by using the features you select and then printing them in colors of your choice.

  2. Differentiation of Cirsium japonicum and C. setosum by TLC and HPLC-MS.

    PubMed

    Ganzera, Markus; Pöcher, Astrid; Stuppner, Hermann

    2005-01-01

    The Chinese Pharmacopoeia indicates the use of field thistle (Cirsium setosum) and Japanese field thistle (C. japonicum) in the treatment of bleeding and inflammation. In the absence of an analytical method for the differentiation and analysis of these two species, TLC and HPLC-MS methods have been developed for this purpose. Both species could be readily distinguished by their flavonoid pattern as revealed by TLC on silica gel layers eluted with ethyl acetate:formic acid:acetic acid:water. The quantitative determination of four flavonoids, namely hispidulin-7-neohesperidoside, linarin, pectolinarin and luteolin, was possible using HPLC. Their optimum separation was achieved on a C12 column eluted with water and 0.025% trifluoroacetic acid in acetonitrile. HPLC-MS experiments were performed to confirm peak identity. In samples of C. japonicum, pectolinarin was the major flavonoid (0.32-2.00%), followed by linarin, hispidulin-7-neohesperidoside and luteolin; the total flavonoid content varied from 0.81 to 3.67%. In C. setosum only one flavonoid (linarin; 1.36-2.83%) was assignable. The HPLC method was validated for linearity, limit of detection (< or = 1.7 ng on-column), peak purity, repeatability (< or = 2.3%) and accuracy (recovery rates of spiked samples were between 99.2 and 101.6%).

  3. HPLC pigment analysis of marine phytoplankton during a red tide occurrence in Tolo Harbour, Hong Kong.

    PubMed

    Wong, C Kwan; Wong, C Kim

    2003-09-01

    A red tide was detected in the inner parts of Tolo Harbour, Hong Kong, in November 2000. Water samples were collected from a fixed station at the centre of the red tide patch for microscopic analysis of phytoplankton community composition and high performance liquid chromatography (HPLC) analysis of phytoplankton pigments. At the peak of the red tide on 24 November 2000, phytoplankton was dominated by the dinoflagellate Scrippsiella trochoidea. The red tide began to decline at the end of November and, by 1 December 2000, the phytoplankton was dominated by diatoms. Chlorophylls and carotenoids in water samples were analysed using HPLC pigment separation technique. Dinoflagellates were indicated by the signature pigment peridinin. Significant correlation (r=0.999) was found between the peridinin concentration and dinoflagellate density. A decrease in peridinin and an increase in fucoxanthin, a major carotenoid in diatoms, marked the shift in phytoplankton composition at the end of the red tide. HPLC analysis also revealed the occurrence of minor phytoplankton groups that are difficult to identify by light microscopy. Red tide monitoring and study of red tide dynamics in Hong Kong have been based on cell counting and spectrophotometric or fluorometric measurement of chlorophyll a. HPLC pigment analysis provides an effective alternative for investigating phytoplankton dynamics during red tide and other algal blooms.

  4. Energy in density gradient

    SciTech Connect

    Vranjes, J.; Kono, M.

    2015-01-15

    Inhomogeneous plasmas and fluids contain energy stored in inhomogeneity and they naturally tend to relax into lower energy states by developing instabilities or by diffusion. But the actual amount of energy in such inhomogeneities has remained unknown. In the present work, the amount of energy stored in a density gradient is calculated for several specific density profiles in a cylindrical configuration. This is of practical importance for drift wave instability in various plasmas, and, in particular, in its application in models dealing with the heating of solar corona because the instability is accompanied with stochastic heating, so the energy contained in inhomogeneity is effectively transformed into heat. It is shown that even for a rather moderate increase of the density at the axis in magnetic structures in the corona by a factor 1.5 or 3, the amount of excess energy per unit volume stored in such a density gradient becomes several orders of magnitude greater than the amount of total energy losses per unit volume (per second) in quiet regions in the corona. Consequently, within the life-time of a magnetic structure such energy losses can easily be compensated by the stochastic drift wave heating.

  5. An Investigation Into HPLC Data Quality Problems

    NASA Technical Reports Server (NTRS)

    Hooker, Stanford B.; VanHeukelem, Laurie

    2011-01-01

    This report summarizes the analyses and results produced by a five-member investigative team of Government, university, and industry experts, established by NASA HQ. The team examined data quality problems associated with high performance liquid chromatography (HPLC) analyses of pigment concentrations in seawater samples produced by the San Diego State University (SDSU) Center for Hydro-Optics and Remote Sensing (CHORS). This report shows CHORS did not validate the methods used before placing them into service to analyze field samples for NASA principal investigators (PIs), even though the HPLC literature contained easily accessible method validation procedures, and the importance of implementing them, more than a decade ago. In addition, there were so many sources of significant variance in the CHORS methodologies, that the HPLC system rarely operated within performance criteria capable of producing the requisite data quality. It is the recommendation of the investigative team to a) not correct the data, b) make all the data that was temporarily sequestered available for scientific use, and c) label the affected data with an appropriate warning, e.g., "These data are not validated and should not be used as the sole basis for a scientific result, conclusion, or hypothesis--independent corroborating evidence is required."

  6. Chromatographically separable rotamers of an unhindered amide

    PubMed Central

    Geffe, Mario; Andernach, Lars; Trapp, Oliver

    2014-01-01

    Summary Surprisingly stable formamide rotamers were encountered in the tetrahydroisoquinoline and morphinan series of alkaloids. We investigated the hindered rotation around the amide bond by dynamic high-performance liquid chromatography (DHPLC) and kinetic measurements of the interconversion of the rotamers which can readily be separated by HPLC as well as TLC. The experimental results of the different methods were compared to each other as well as to results obtained by DFT calculations. PMID:24778722

  7. Magnetic Separation Dynamics of Colloidal Magnetic Nanoparticles

    SciTech Connect

    Kaur, M.; Huijin Zhang,; You Qiang,

    2013-01-01

    Surface functionalized magnetic nanoparticles (MNPs) are appealing candidates for analytical separation of heavy metal ions from waste water and separation of actinides from spent nuclear fuel. This work studies the separation dynamics and investigates the appropriate magnetic-field gradients. A dynamic study of colloidal MNPs was performed for steady-state flow. Measurements were conducted to record the separation time of particles as a function of magnetic field gradient. The drag and magnetic forces play a significant role on the separation time. A drop in saturation magnetization and variation of particle size occurs after surface functionalization of the MNPs; these are the primary factors that affect the separation time and velocity of the MNPs. The experimental results are correlated to a theoretical one-dimensional model.

  8. Isotope separation

    DOEpatents

    Bartlett, Rodney J.; Morrey, John R.

    1978-01-01

    A method and apparatus is described for separating gas molecules containing one isotope of an element from gas molecules containing other isotopes of the same element in which all of the molecules of the gas are at the same electronic state in their ground state. Gas molecules in a gas stream containing one of the isotopes are selectively excited to a different electronic state while leaving the other gas molecules in their original ground state. Gas molecules containing one of the isotopes are then deflected from the other gas molecules in the stream and thus physically separated.

  9. ISOTOPE SEPARATORS

    DOEpatents

    Bacon, C.G.

    1958-08-26

    An improvement is presented in the structure of an isotope separation apparatus and, in particular, is concerned with a magnetically operated shutter associated with a window which is provided for the purpose of enabling the operator to view the processes going on within the interior of the apparatus. The shutier is mounted to close under the force of gravity in the absence of any other force. By closing an electrical circuit to a coil mouated on the shutter the magnetic field of the isotope separating apparatus coacts with the magnetic field of the coil to force the shutter to the open position.

  10. Development of a Single Ion Pair HPLC Method for Analysis of Terbinafine, Ofloxacin, Ornidazole, Clobetasol, and Two Preservatives in a Cream Formulation: Application to In Vitro Drug Release in Topical Simulated Media-Phosphate Buffer Through Rat Skin.

    PubMed

    Dewani, Anil P; Bakal, Ravindra L; Kokate, Pranjali G; Chandewar, Anil V; Patra, Srdhanjali

    2015-01-01

    Present work reports an HPLC method with UV detection for quantification of terbinafine, ofloxacin, ornidazole, and clobetasol in a cream formulation along with two preservatives methyl and propyl paraben. The chromatographic separation and quantification was achieved by an octyl bonded column and a gradient elution program involving an ion-pairing reagent, hexanesulfonic acid (0.2%, pH modified to 2.7 using orthophosphoric acid) and acetonitrile. The method was simple and devoid of buffer salts and therefore advantageous for system and column life. The three step gradient program was initiated with 30% (v/v) acetonitrile for the first 5 min and ramped linearly to 60% in the next 7 min. The mobile phase remained constant for the next 11 min and then concluded at 30% (v/v) of acetonitrile. Flow rate throughout was 0.8 mL/min, and all the signals were monitored at 243 nm. The method was applied for assay of a cream formulation and its in vitro permeation studies to determine the penetration profile of the four drugs and two preservatives. A marketed cream formulation was selected for the permeation study, which was carried out using a diffusion cell consisting of topical simulated media, phosphate buffer (pH=6.8) solution containing 1% sodium lauryl sulfate as a receiver medium.

  11. SEPARATION PROCESS

    DOEpatents

    Stoughton, R.W.

    1961-10-24

    A process for separating tetravalent plutonium from aqueous solutions and from niobium and zirconium by precipitation on lanthanum oxalate is described. The oxalate ions of the precipitate may be decomposed by heating in the presence of an oxidizing agent, forming a plutonium compound readily soluble in acid. (AEC)

  12. Plasma separation

    NASA Technical Reports Server (NTRS)

    Steurer, Wolfgang

    1992-01-01

    This process employs a thermal plasma for the separation and production of oxygen and metals. It is a continuous process that requires no consumables and relies entirely on space resources. The almost complete absence of waste renders it relatively clean. It can be turned on or off without any undesirable side effects or residues. The prime disadvantage is its high power consumption.

  13. Status and use of HPLC-DAD/APCI-MS and direct flame sampling/APCI-MS for fullerenes and PAH research. First annual technical report for instrumentation grant, Project period 1 August 1992--31 July 1994

    SciTech Connect

    Howard, J.B.; Lafleur, A.L.; Taghizadeh, K.

    1994-08-01

    The PE/Sciex APCI/LC-MS equipment consists of a high performance liquid chromatograph (HPLC) with diode array spectrophotometric detector (DAD) interfaced to an atmospheric pressure ionizer with a single quadrupole mass spectrometer (MS) through a heated nebulizer. The interface between the HPLC and the MS is designed to minimize thermal input to the HPLC eluents and is of great importance for the authors intended application. Ionization occurs in a unique fashion at atmospheric pressure using a chemical ionization source (APCI). The equipment is being used for the identification and quantification of flame-generated fullerenes and polycyclic aromatic hydrocarbons (PAH). The mode of operation of this instrument is as follows: (1) complex mixtures are separated by the HPLC; (2) UV spectra of separated components are obtained by the diode array detector (DAD) as they elute from the HPLC; (3) components undergo mild ionization in the APCI source; and, (4) mass spectra are acquired by the mass spectrometer.

  14. Non Linear Conjugate Gradient

    2006-11-17

    Software that simulates and inverts electromagnetic field data for subsurface electrical properties (electrical conductivity) of geological media. The software treats data produced by a time harmonic source field excitation arising from the following antenna geometery: loops and grounded bipoles, as well as point electric and magnetic dioples. The inversion process is carried out using a non-linear conjugate gradient optimization scheme, which minimizes the misfit between field data and model data using a least squares criteria.more » The software is an upgrade from the code NLCGCS_MP ver 1.0. The upgrade includes the following components: Incorporation of new 1 D field sourcing routines to more accurately simulate the 3D electromagnetic field for arbitrary geologic& media, treatment for generalized finite length transmitting antenna geometry (antennas with vertical and horizontal component directions). In addition, the software has been upgraded to treat transverse anisotropy in electrical conductivity.« less

  15. Generalized conjugate gradient squared

    SciTech Connect

    Fokkema, D.R.; Sleijpen, G.L.G.

    1994-12-31

    In order to solve non-symmetric linear systems of equations, the Conjugate Gradient Squared (CGS) is a well-known and widely used iterative method. In practice the method converges fast, often twice as fast as the Bi-Conjugate Gradient method. This is what you may expect, since CGS uses the square of the BiCG polynomial. However, CGS may suffer from its erratic convergence behavior. The method may diverge or the approximate solution may be inaccurate. BiCGSTAB uses the BiCG polynomial and a product of linear factors in an attempt to smoothen the convergence. In many cases, this has proven to be very effective. Unfortunately, the convergence of BiCGSTAB may stall when a linear factor (nearly) degenerates. BiCGstab({ell}) is designed to overcome this degeneration of linear factors. It generalizes BiCGSTAB and uses both the BiCG polynomial and a product of higher order factors. Still, CGS may converge faster than BiCGSTAB or BiCGstab({ell}). So instead of using a product of linear or higher order factors, it may be worthwhile to look for other polynomials. Since the BiCG polynomial is based on a three term recursion, a natural choice would be a polynomial based on another three term recursion. Possibly, a suitable choice of recursion coefficients would result in method that converges faster or as fast as CGS, but less erratic. It turns out that an algorithm for such a method can easily be formulated. One particular choice for the recursion coefficients leads to CGS. Therefore one could call this algorithm generalized CGS. Another choice for the recursion coefficients leads to BiCGSTAB. It is therefore possible to mix linear factors and some polynomial based on a three term recursion. This way one may get the best of both worlds. The authors will report on their findings.

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

    NASA Astrophysics Data System (ADS)

    Brenneman, Charles A.; Ebeler, Susan E.

    1999-12-01

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

  17. Enantioselective determination of metoprolol and its metabolites in human urine high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and tandem mass spectrometry (MS/MS).

    PubMed

    Baranowska, Irena; Adolf, Weronika; Magiera, Sylwia

    2015-11-01

    A sensitive, stereoselective assay using solid phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) was developed and validated for the analysis of enantiomers of metoprolol and its metabolites (α-hydroxymetoprolol, O-desmethylmetoprolol). Chiral separation was achieved using a CHIRALCEL OD-RH column, packed with cellulose tris-(3,5-dimethylphenyl-carbamate) stationary phase, employing a mobile phase composed by a mixture of 0.2% diethylamine in water and acetonitrile in gradient elution mode. Linear calibration curves were obtained over the range of 0.025-2.0μg/mL (R(2)>0.994) in urine for both enantiomers of metoprolol and its metabolites with quantitation limit of 0.025μg/mL. Intra and inter-day precision and accuracy were below 15% for both metoprolol and metabolites enantiomers. The recovery of enantiomer of metoprolol and its metabolite was greater than 68.0%, utilizing a SPE procedure. The method was tested with urine quality control samples and human urine fractions after administration of 50mg rac-metoprolol.

  18. Quantification of tetramethyl-terephthalic acid in rat liver, spleen and urine matrices by liquid-liquid phase extraction and HPLC-photodiode array detection.

    PubMed

    Baati, Tarek; Horcajada, Patricia; David, Olivier; Gref, Ruxandra; Couvreur, Patrick; Serre, Christian

    2012-01-01

    Tetramethyl-terephthalate (TMT) is the constitutive linker of the flexible porous iron(III) carboxylate Metal Organic Framework (MOF) MIL-88B_4CH₃ based drug nanocarrier (MIL stands for Material from Institut Lavoisier). A method for the determination of the concentration of tetramethyl-terephthalic acid has been developed in different biological rat matrices (liver, spleen and urine) using a liquid-liquid phase extraction and high-performance liquid chromatography (HPLC) coupled to photodiode array detection with 4-aminosalicylic acid as internal standard. The extraction conditions of TMT have been varied from urine to tissue depending on the complexity of the biological matrices. The chromatographic separation was performed with a gradient elution. In all matrices, the limits of detection and quantification of TMT was 0.01 and 0.05 μg ml⁻¹, respectively. The recovery of the TMT reached 86, 89 and 97% for urine, spleen and liver tissues, respectively. The linearity of the calibration curves in urine and tissues was satisfactory in all cases as evidenced by correlation coefficients >0.990. The within-day and between-day precisions were <15% (n=6) and the accuracy ranged in all cases between 86 and 103%. This method has finally allowed the quantification of TMT in rat urine and in tissue samples of rats administered intravenously with iron(III) tetramethyltherepthalate MIL-88B_4CH₃ nanoparticles.

  19. Identification and Simultaneous Determination of Twelve Active Components in the Methanol Extract of Traditional Medicine Weichang’an Pill by HPLC-DAD-ESI-MS/MS

    PubMed Central

    Zhang, Jingze; Gao, Wenyuan; Liu, Zhen; Zhang, Zhidan

    2013-01-01

    Weichang’an (WCA) pill, a traditional Chinese patent medicine consisting of ten Chinese medicinal herbs, has been used to treat irritable bowel syndrome and functional dyspepsia for several decades. In this study, twelve bioactive constituents in the methanol extract of WCA were accurately identified since MS/MS fragmentation behavior of the references and the standards by using HPLC-DAD-ESI-MS/MS analysis and a reliable and accurate method for the simultaneous determination was developed. Twelve active components including costunolide and dehydrodehydrocostus lactone from the principal herb Radix Aucklandiae; naringin, hesperidin and neohesperidin from Fructus Aurantii; magnolol and honokiol from the ministerial herbs Cortex Magnoliae officinalis; aloe-emodin, rhein, emodin, chrysophanol and physcion from adjunctive and messenger herb Radix et Rhizoma Rhei were analyzed in the samples. The chromatographic separation was performed on a Kromasil C18 column with gradient elution of acetonitrile-methanol and 1.0% acetic acid water. In this condition, linearity, inter- and intra-day precision and accuracy were within acceptable ranges. The developed method showed satisfactory precision and accuracy with overall intra- and inter-day variations of 0.68-1.33% and 0.67-2.05% respectively, and the overall recoveries of 97.54-102.69% for twelve compounds. The proposed approach was successfully applied as a powerful tool for the quality control of WCA pill. PMID:24250567

  20. An HPLC-MS/MS method for simultaneous determination of the active metabolites of febuxostat (67M-1, 67M-2 and 67M-4) in human plasma.

    PubMed

    Xie, Huiru; Wang, Zhijun; Deng, Ke; Jiang, Xuehua; Wang, Ling; Lv, Guoyu

    2014-11-01

    An HPLC-MS/MS method for simultaneously determination of the active metabolites (67M-1, 67M-2 and 67M-4) in human plasma using clopidogrel as the internal standard was developed and validated. The compounds were extracted by protein precipitation using acetonitrile and separated using a C8 column by a gradient elution with the mobile phase consisting of acetonitrile (containing 0.1% formic acid) and 0.1% formic acid. Quantification was performed using multiple reaction monitoring in positive mode with m/z transitions of 333.1-261.0, 333.1-261.0, 347.0-261.0 and 322.2-184.1 for 67M-1, 67M-2, 67M-4 and clopidogrel (Internal Standard), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The lower limit of quantification of this method was 0.5 ng/mL and the calibration curve was linear over the concentration range of 0.5-150 ng/mL. The intra- and inter-run precision was less than 11.67% and 8.64%, respectively, with the accuracy between 98.33% and 108.38%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study of febuxostat in healthy Chinese volunteers following oral administration of 40 mg and 80 mg febuxostat.

  1. Novel methodology for the extraction and identification of natural dyestuffs in historical textiles by HPLC-UV-Vis-ESI MS. Case study: chasubles from the Wawel Cathedral collection.

    PubMed

    Lech, Katarzyna; Jarosz, Maciej

    2011-03-01

    High-performance liquid chromatography coupled with spectrophotometric and electrospray mass spectrometric detection (HPLC-UV-Vis-ESI MS) was used for characterization of natural dyes present in historical art works. The gradient program was developed for identification of 29 colorants of various polarities. Dual detection system (UV-Vis and ESI MS) allowed differentiation of all compounds, even if they were not completely separated. This enabled examination of more color compounds over a substantially shorter time in comparison with previously recommended methods. Moreover, for extraction of colorants from historical textiles a two-step sequential procedure was proposed, excluding evaporation used in earlier procedures. The developed method was successfully applied to identification of indigotin, carminic, kermesic, flavokermesic, dcII, dcIV, dcVII, and ellagic acids as well as luteolin, apigenin, and genistein in red, violet, and green fibers taken from three selected historical chasubles which belong to the collection of the Wawel Cathedral treasury (Cracow, Poland). Italian textiles from the fifteenth and sixteenth centuries, of which chasubles were made, were dyed with a limited number of dyestuffs, consistently used for all batches of fabrics. The obtained results also allowed confirmation of the structure of the so-called "dcII" component of cochineal as a C-glucose derivative of flavokermesic acid. PMID:21188578

  2. Design of Experiment (DOE) Utilization to Develop a Simple and Robust Reversed-Phase HPLC Technique for Related Substances’ Estimation of Omeprazole Formulations

    PubMed Central

    Manranjan, Vayeda Chintan; Yadav, Devendra Singh; Jogia, Hitesh Amrutlal; Chauhan, Praful Lalitkumar

    2013-01-01

    A simple, fast, and sensitive reversed-phase HPLC method with UV detection was developed for the quantitation of omeprazole and its eleven related compounds (impurities) in pharmaceutical formulation using the Thermo Accucore C–18 (50 mm × 4.6 mm, 2.6 μm) column. The separation among all the compounds was achieved with a flow rate of 0.8 mL min−1 employing a gradient program of mobile phase A [0.08 M glycine buffer pH 9.0: acetonitrile; 95:05 (v/v)] and mobile phase B [acetonitrile: methanol; 65:35 (v/v)]. The chromatographic detection was carried out at a wavelength of 305 nm. The method was validated for specificity, linearity, and recovery. The huskiness of the method was determined prior to validation using the Design of Experiments (DOE). The ANOVA analysis of DOE with a 95% confidence interval (CI) confirmed the buffer pH of mobile phase A (p <0.0001) and column temperature (p<0.0001) as significant Critical Method Parameters (CMPs). PMID:24482772

  3. Development of HPLC method by UV-VIS detection for the quantification of phenolic acids in different Ocimum sanctum Linn. extracts.

    PubMed

    Shafqatullah; Khan, Rasool; Hassan, Waseem; Hussain, Arshad; Asadullah; Rehman, Khaliqur; Ali, Javid

    2014-09-01

    A simple and rapid chromatographic method has been developed for the simultaneous determination of five phenolic acids including Gallic acid, Chloroganic acid, Syringic acid, Benzoic acid and Vanillic acid by HPLC with UV-VIS detector. These Phenolic acids were separated by analytical column Intersil ODS-3 C18, a gradient elution system of ACN and acidified water solution with 1ml/min flow rate and quantified in a total run of 30 minutes at 210nm wavelength. In the quantitative analysis of these compounds showed good regression (0.995-0.999). The limit of detection [LOD] and limit of quantification [LOQ] of these compounds were in the range of 0.15-0.46 and 0.42-2.47 βg/mL. The average recoveries were between 95.8-103.1% and their RSD values were less than 3.34%. By the proposed method Gallic acid, Chloroganic acid and Syringic acid were found and quantified in Methanolic, Ethanolic and Acetonic extract of Ocimum sanctum Linn. leaves. While the two other phenolic acids benzoic acid and vanillic acid was not found in the extracts of Ocimum sanctum Linn. leaves. PMID:25176382

  4. Simultaneous determination of nine coumarins in rat plasma by HPLC-MS/MS for pharmacokinetics studies following oral administration of Fraxini Cortex extract.

    PubMed

    Zhao, Minmin; Ding, Weijing; Wang, Shuang; Wang, Chunying; Du, Yingfeng; Xu, Huijun; Wang, Qiao; Jin, Shumin

    2016-07-01

    A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous determination of nine coumarins including aesculin, aesculetin, fraxin, fraxetin, scopoletin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin, 8-hydroxy-6,7-dimethoxy coumarin and umbelliferone in rat plasma using nodakenin as the internal standard (IS). The plasma samples were pretreated by a one-step direct protein precipitation with methanol. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of methanol and water (containing 0.05% acetic acid). All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. This method was fully validated in terms of the sensitivity, specificity, accuracy, precision (intra- and inter-day), matrix effect, recovery as well as the stability of the analyte under various conditions, and the results satisfied the requirements of biological sample measurement. The validated method was successfully applied to pharmacokinetic study of the nine coumarins in rat plasma after oral administration of Fraxini Cortex aqueous extract, among which the pharmacokinetics of four coumarins including fraxetin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin and 8-hydroxy-6,7-dimethoxy coumarin were studied for the first time.

  5. Microwave-assisted extraction in combination with HPLC-UV for quantitative analysis of six bioactive oxoisoaporphine alkaloids in Menispermum dauricum DC.

    PubMed

    Wei, Jinxia; Chen, Jing; Liang, Xinlei; Guo, Xingjie

    2016-02-01

    A novel and reliable method based on microwave-assisted extraction (MAE) followed by HPLC-UV was developed and validated for the simultaneous quantification of six pharmacologically important oxoisoaporphine alkaloids in the total plants of Menispermum dauricum DC. The optimal MAE extraction condition was performed at 60°C for 11 min with ethanol-water (70:30, v/v) as the extracting solvent, and the solvent to solid ratio was 20:1. Chromatographic separation was achieved on a reversed-phase YMC C18 column (250 × 4.6 mm, i.d., 5 µm) with a gradient mobile phase consisting of A (1% aqueous formic acid) and B (acetonitrile containing 1% formic acid) at a flow rate of 1.5 mL/min. The detection wavelength was set at 422 nm. Excellent linearity over the investigated concentration ranges was observed with values of r >0.999 for all analytes. The method developed was validated with acceptable sensitivity, intra- and inter-day precision and extraction recoveries. It was successfully applied to the determination of six alkaloids in Menispermum dauricum DC from different sources and different parts of Menispermum dauricum DC. The results obtained indicated that the method is suitable for the quality control of Menispermum dauricum DC.

  6. Simultaneous determination and pharmacokinetic study of water-soluble and lipid-soluble components of danshen in rat plasma using HPLC-UV method.

    PubMed

    Wang, Xiuli; Zhang, Zhi-Rong; Fu, Hualin; Liu, Jie; Chen, Qian; Nie, Yu; Deng, Li; Gong, Tao

    2007-11-01

    A sensitive and specific HPLC-UV method was developed for the simultaneous determination of major active components of danshen in rat plasma. Both water-soluble and lipid-soluble compounds were included, i.e. danshensu, salvianolic acid B and tanshinone IIA. Protocatechuic aldehyde and diazepam were used as internal standards. The chromatographic separation was achieved on a reversed-phase C(18) column by gradient elution using acetonitrile and 0.025% (v/v) phosphoric acid solution as mobile phase, at a flow rate of 1.0 mL/min. Salvianolic acid B, danshensu and internal standards were detected at 281 nm, while the detection of tanshinone IIA was carried out at 272 nm. All calibration curves showed good linearity (r(2) > 0.999) within test ranges. The limit of detection and the limit of quantification for danshensu, salvianolic acid B and tanshinone IIA in plasma were 0.065, 0.043, 0.022, 0.131, 0.085 and 0.044 microg/mL, respectively. This is the first report on the determination and pharmacokinetic study of danshensu, salvianolic acid B and tanshinone IIA in rat plasma and the results indicated that this method was reliable for the determination of the major active components of danshen in rat plasma. PMID:17685392

  7. Development and validation of an HPLC-DAD analytical procedure for quality control of damiana (Turnera diffusa), using an antioxidant marker isolated from the plant.

    PubMed

    Pérez-Meseguer, Jonathan; Garza-Juárez, Aurora; Salazar-Aranda, Ricardo; Salazar-Cavazos, Ma Luz; de la Torre Rodríguez, Yael C; Rivas-Galindo, Verónica; Waksman de Torres, Noemí

    2010-01-01

    Damiana is a shrub used as a dietary supplement and as an additive in several foods. This report presents the first validated analytical method for QC of products made from damiana. Through bioassay-guided fractionation, the compound showing the best antioxidant activity, determined using 2,2-diphenyl-1-picryl-hydrazyl assay in TLC, was purified and identified as 8-C-beta-[6-deoxy-2-O-(alpha-1-rhamnopyranosyl)-xylo- hexopyranos-3-uloside] (Cl50 5.56 g/mL). This is the only natural source from which this compound has been reported, and has been found in all native damiana samples analyzed. Therefore, it was used as a biomarker to develop an HPLC-diode-array detection analytical procedure using the first-order derivative of the chromatograms. Separation was achieved using an AccQ Tag C18 150 x 3.9 mm (4 microm) column, with a gradient of methanol and 0.1% trifluoroacetic acid in water. The method was shown to be accurate, sensitive, and reproducible, providing a useful quantitative means to apply QC to damiana products. PMID:20922948

  8. Microwave-assisted extraction in combination with HPLC-UV for quantitative analysis of six bioactive oxoisoaporphine alkaloids in Menispermum dauricum DC.

    PubMed

    Wei, Jinxia; Chen, Jing; Liang, Xinlei; Guo, Xingjie

    2016-02-01

    A novel and reliable method based on microwave-assisted extraction (MAE) followed by HPLC-UV was developed and validated for the simultaneous quantification of six pharmacologically important oxoisoaporphine alkaloids in the total plants of Menispermum dauricum DC. The optimal MAE extraction condition was performed at 60°C for 11 min with ethanol-water (70:30, v/v) as the extracting solvent, and the solvent to solid ratio was 20:1. Chromatographic separation was achieved on a reversed-phase YMC C18 column (250 × 4.6 mm, i.d., 5 µm) with a gradient mobile phase consisting of A (1% aqueous formic acid) and B (acetonitrile containing 1% formic acid) at a flow rate of 1.5 mL/min. The detection wavelength was set at 422 nm. Excellent linearity over the investigated concentration ranges was observed with values of r >0.999 for all analytes. The method developed was validated with acceptable sensitivity, intra- and inter-day precision and extraction recoveries. It was successfully applied to the determination of six alkaloids in Menispermum dauricum DC from different sources and different parts of Menispermum dauricum DC. The results obtained indicated that the method is suitable for the quality control of Menispermum dauricum DC. PMID:26094998

  9. Determination of copper, nickel, cobalt, silver, lead, cadmium, and mercury ions in water by solid-phase extraction and the RP-HPLC with UV-Vis detection.

    PubMed

    Hu, Qiufen; Yang, Guangyu; Zhao, Yiyun; Yin, Jiayuan

    2003-03-01

    A new method for the simultaneous determination of seven heavy metal ions in water by solid-phase extraction and reversed-phase high-performance liquid chromatography (RP-HPLC) was developed. The copper, nickel, cobalt, silver, lead, cadmium, and mercury ions were pre-column derivatized with tetra( m-aminophenyl)porphyrin (T m-APP) to form colored chelates. The metal-T m-APP chelates in 100 mL of sample were preconcentrated to 1 mL by solid-phase extraction with a C(18 )cartridge; an enrichment factor of 100 was achieved. The chelates were separated on a Waters Xterra()RP(18) column by gradient elution with methanol (containing 0.05 mol L(-1) pyrrolidine-acetic acid buffer salt, pH 10.0) and acetone (containing 0.05 mol L(-1) pyrrolidine-acetic acid buffer salt, pH 10.0) as mobile phase at a flow rate of 1.0 mL min(-1) and detected with a photodiode array detector. The detection limits of copper, cobalt, nickel, silver, lead, cadmium, and mercury are 2, 2, 3, 4, 3, 3, and 3 ng L(-1), respectively, in the original sample. The method was also applied to the determination of these metals in water with good results. PMID:12664186

  10. Novel Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Clindamycin Phosphate and Adapalene along with Preservatives in Topical Gel Formulations.

    PubMed

    Modi, Prakash B; Shah, Nehal J

    2014-12-01

    A novel stability-indicating RP-HPLC method was developed for the simultaneous estimation of clindamycin phosphate (hydrophilic), adapalene (hydro-phobic), phenoxyethanol, and methylparaben in topical gel formulations. Optimum chromatographic separation among the analytes and stress-induced degradants peaks was achieved on the XBridge C18 (50 × 4.6 mm, 3.5 µm) column using a mobile phase consisting of a variable mixture of pH 2.50 ammonium hydrogen phosphate buffer, acetonitrile, and tetrahydrofuran with gradient elution. Detection was performed at 210 nm for phenoxyethanol, methylparaben, and clindamycin phosphate and 321 nm for adapalene. The method was optimized with a unique diluent selection for the extraction of clindamycin phosphate and adapalene from the gel matrix. The developed method was validated for method precision, specificity, LOD and LOQ, linearity, accuracy, robustness, and solution stability as per ICH guidelines. The proposed method can be employed for the quantification of clindamycin phosphate, adapalene, phenoxyethanol, and methylparaben in commercial topical gel formulations.

  11. Determination of chlorpheniramine in human plasma by HPLC-ESI-MS/MS: application to a dexchlorpheniramine comparative bioavailability study.

    PubMed

    Moreno, Ronilson Agnaldo; Oliveira-Silva, Diogo; Sverdloff, Carlos Eduardo; Borges, Bruno Carter; Rebelo Galvinas, Paulo Alexandre; Astigarraga, Rafael Barrientos; Borges, Ney Carter

    2010-07-01

    In the present study a fast, sensitive and robust validated method to quantify chlorpheniramine in human plasma using brompheniramine as internal standard (IS) is described. The analyte and the IS were extracted from plasma by LLE (diethyl ether-dichloromethane, 80:20, v/v) and analyzed by HPLC-ESI-MS/MS. Chromatographic separation was performed using a gradient of methanol from 35 to 90% with 2.5 mm NH(4)OH on a Gemini Phenomenex C(8) 5 microm column (50 x 4.6 mm i.d.) in 5.0 min/run. The method fitted to a linear calibration curve (0.05-10 ng/mL, R > 0.9991). The precision (%CV) and accuracy ranged, respectively: intra-batch from 1.5 to 6.8% and 99.1 to 106.6%, and inter-batch from 2.4 to 9.0%, and 99.9 to 103.1%. The validated bioanalytical procedure was used to assess the comparative bioavailability in healthy volunteers of two dexchlorpheniramine 2.0 mg tablet formulations (test dexchlorpheniramine, Eurofarma, and reference Celestamine, Schering-Plough). The study was conducted using an open, randomized, two-period crossover design with a 2 week washout interval. Since the 90% confidence interval for C(max) and AUC ratios were all within the 80-125% interval proposed by ANVISA and FDA, it was concluded that test and reference formulations are bioequivalent concerning the rate and the extent of absorption.

  12. A new strategy for quality control and qualitative analysis of Yinhuang preparations by HPLC-DAD-MS/MS.

    PubMed

    Chen, Hui; Chen, Xu; Han, Qiang; Wu, Jing; Tang, Dao-quan; Du, Qian; Yin, Xiao-xing; Yang, Dong-zhi

    2012-10-01

    A combinative method using fingerprint analysis (FA) and multi-ingredients quantification (MIQ) was developed and validated for the quality control of Yinhuang (YH) preparations including granule, capsule, and lozenge by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). Common peaks with or without standard references in FA were confirmed or identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The chromatographic separations were achieved on a Sepax GP-C(18) column (250 mm × 4.6 mm i.d., 5 μm) with a gradient elution using a mixture of 0.1 % formic acid methanol solution and 0.1 % formic acid water solution. In quantitative analysis, nine bioactive constituents (chlorogenic acid, caffeic acid, luteoloside, baicalin, luteolin, wogonoside, baicalein, wogonin, and oroxylin A) were simultaneously determined. The detection wavelength was set at 275 nm, 320 nm, and 350 nm according to the absorption properties of the nine quantified compounds. The linearity, recovery, intraday and interday precision, accuracy, limit of detection (LOD) and quantification (LOQ), repeatability and stability were all tested and good results were obtained. In the FA, 320 nm was selected. The correlation coefficients of similarity were determined on the basis of the relative retention time (RRT) and relative peak area (RPA) of 20 common peaks in chromatographic fingerprints. Results indicated that both the RRT and RPA of 20 common peaks shared a close similarity. From the 20 common peaks, 18 compounds, including the nine quantified compounds, were identified or tentatively characterized by comparing their retention times, UV spectra, and MS spectra with those of standard compounds or literature data. The study not only presents a powerful and reliable analytical tool for the quality control of YH preparations, but also provides the chemical evidence for revealing the material basis of their therapeutic effects.

  13. A new strategy for quality control and qualitative analysis of Yinhuang preparations by HPLC-DAD-MS/MS.

    PubMed

    Chen, Hui; Chen, Xu; Han, Qiang; Wu, Jing; Tang, Dao-quan; Du, Qian; Yin, Xiao-xing; Yang, Dong-zhi

    2012-10-01

    A combinative method using fingerprint analysis (FA) and multi-ingredients quantification (MIQ) was developed and validated for the quality control of Yinhuang (YH) preparations including granule, capsule, and lozenge by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). Common peaks with or without standard references in FA were confirmed or identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The chromatographic separations were achieved on a Sepax GP-C(18) column (250 mm × 4.6 mm i.d., 5 μm) with a gradient elution using a mixture of 0.1 % formic acid methanol solution and 0.1 % formic acid water solution. In quantitative analysis, nine bioactive constituents (chlorogenic acid, caffeic acid, luteoloside, baicalin, luteolin, wogonoside, baicalein, wogonin, and oroxylin A) were simultaneously determined. The detection wavelength was set at 275 nm, 320 nm, and 350 nm according to the absorption properties of the nine quantified compounds. The linearity, recovery, intraday and interday precision, accuracy, limit of detection (LOD) and quantification (LOQ), repeatability and stability were all tested and good results were obtained. In the FA, 320 nm was selected. The correlation coefficients of similarity were determined on the basis of the relative retention time (RRT) and relative peak area (RPA) of 20 common peaks in chromatographic fingerprints. Results indicated that both the RRT and RPA of 20 common peaks shared a close similarity. From the 20 common peaks, 18 compounds, including the nine quantified compounds, were identified or tentatively characterized by comparing their retention times, UV spectra, and MS spectra with those of standard compounds or literature data. The study not only presents a powerful and reliable analytical tool for the quality control of YH preparations, but also provides the chemical evidence for revealing the material basis of their therapeutic effects. PMID:22885972

  14. [Determination of myclobutanil 25% WG degradation dynamics in ginseng root, stem, leaf and soil by HPLC-MS/MS].

    PubMed

    Wang, Yan; Wang, Chun-Wei; Gao, Jie; Cui, Li-Li; Xu, Yun-Cheng

    2014-07-01

    A high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed for determining degradation dynamics and final residues of myclobutanil 25% WG in ginseng root, stem, leaf and soil. The samples were extracted with acetonitrile, cleaned-up with primary secondary amine (PSA) solid phase extraction cartridge, separated by Kromasil Eternity-5-C18 (2.1 mm x 150 mm, 5 microm) column with a gradient of acetonitrile and 0.1% formate in water as mobile phases, and analyzed with the multiple reaction monitoring (MRM) in positive ion mode by employing the external standard method. The average recoveries and the relative standard derivations (RSDs) of myclobutanil at the spiked level of 0.01-0.20 mg x kg(-1) were 80.9%-90.7% and 5.54%-9.29%, respectively, and the limit of quantification (LOQ) was 0.005 mg x kg(-1). The method with good reproducible, high precision and low detection limit could meet the requirements of residual analysis on ginseng production. The half-lives of myclobutanil were from 6.25 days to 9.94 days in ginseng root, stem, leaf and soil at spraying dosage of 1 152 g x hm(-2) The final residues were below 0.060 1 mg x kg(-1) in root, below 0.081 7 mg x kg(-1) in stem, 0.006 0-0.102 2 mg x kg(-1) in leaf and below 0.037 6 mg x kg(-1) in soil at spraying dosage range from 576 to 1 152 g x hm(-2). It is recommended that the MRLs of myclobutanil in dried ginseng may be suggested to be 0.10 mg x kg(-1) temporarily, and the preharvest interval was set at 35 days.

  15. [Simultaneous determination of five cold medicine ingredients in paracetamol triprolidine hydrochloride and pseudoephedrine hydrochloride tablets by pH/organic solvent double-gradient high performance liquid chromatography].

    PubMed

    Xuan, Xueyi; Huang, Lina; Pan, Xiaoling; Li, Ning

    2013-02-01

    A pH/organic solvent double-gradient mode in reversed-phase high performance liquid chromatography (HPLC) has been established as a new approach to the simultaneous determination of acetaminophen, caffeine, salicylamide, pseudoephedrine hydrochloride and triprolidine hydrochloride in paracetamol triprolidine hydrochloride and pseudoephedrine hydrochloride tablets. Through the optimization of the organic solvent gradient mode and pH/organic solvent double-gradient mode, the optimum double-gradient HPLC system of the five cold medicine ingredients has been built. The determination was carried out on a Diamonsiol C18 column (250 mm x 4.6 mm, 5 microm). The mobile phase consisted of methanol, 0.05 mol/L ammonium acetate solution and 0.08 mol/L acetic acid solution. The column temperature was set at 30 degrees C. The flow rate was 1.0 mL/min. The sample was measured at multiple wavelengths: 0-6 min, 280 nm; 6-7 min, 257 nm; 7-14 min, 280 nm; 14 min, 233 nm. The separation of the five cold medicine ingredients in the tablets was achieved in 25.5 min. The linear ranges of acetaminophen, pseudoephedrine hydrochloride, caffeine, salicylamide and triprolidine hydrochloride were 0.055 -0.998 g/L, 0.053-0.946 g/L, 0.007-0.129 g/L, 0.035-0.622 g/L and 0.002-0.039 g/L, respectively, with their correlation coefficients greater than 0.999 0. The detection limits (S/N = 3) were 0.09, 6, 0.02, 0.128 and 0.02 mg/L, respectively. Their mean recoveries were 97.9%-102.8%. The advantage of the method is the simultaneous determination of acidic, neutral and basic compounds. It also can improve the column efficiency of the analyte, compress the half-peak width and reduce the trailing. The optimized and validated method can be used for the simultaneous determination of the five cold medicine ingredients in the tablets.

  16. Determination of theophylline by HPLC and GC-IDMS, the effect of chemically similar xanthine derivatives on the specificity of the method and the possibility of paracetamol as interfering substance.

    PubMed

    Kress, Michael; Meissner, Dieane; Kaiser, Patricia; Hanke, Rainer; Wood, William Graham

    2002-01-01

    The aim of this study was to develop and compare high-performance liquid chromatography (HPLC) and gas chromatography coupled with isotope dilution-mass spectrometry (GC-IDMS) methods with a common extraction procedure for the determination of substituted xanthines in biological matrices such as serum and urine. For HPLC both isocratic and gradient methods were evaluated. Difficulties occurred in separation of all 6 xanthines of interest - uric acid, theobromine, theophylline, paraxanthine, caffeine and 1,3-dimethyl-7-(2-hydroxyethyl) xanthine as internal standard. In addition, paracetamol was seen to interfere at higher concentrations, which meant that a system had to be developed to separate all 7 components of interest. The final solution chosen consisted of precipitation of serum samples with 6 mol/l trichloroacetic acid followed by neutralisation with 3 mol/l KOH and chromatography on a 150 x 4.6 mm Nautilus C-18 column (Macherey & Nagel) using an isocratic elution consisting of 0.02 mol/l acetate-phosphate buffer, pH 3.0 containing 9.6% v/v acetonitrile and monitoring at 273 +/- 7 nm. Comparisons with GC-IDMS and FPIA were acceptable. Run times of 10 minutes were possible. An additional "safe time" of 5 minutes was allowed to elute any substances with similar absorption maxima which were sometimes present in commercial control sera. Precision of the method was 1.64% (intra-assay) and 2.87% (inter-assay) at 4.1 mg/l and 1.51% respectively 2.15% at 25 mg/l including extraction and measurement steps. Recovery was between 86 and 101% between 1.25 and 100 mg/l and peak time deviations for all 7 components between 0.07% and 0.34% (coefficient of variation) in 7 consecutive measurements.

  17. HPLC of the Polypeptides in a Hydrolyzate of Egg-White Lysozyme. An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Richardson, W. S., III; Burns, L.

    1988-01-01

    Describes a simple high-performance liquid chromatography experiment for undergraduate biochemistry laboratories. The experiment illustrates the separation of polypeptides by a step gradient elution using a single pump instrument with no gradient attachments. Discusses instrumentation, analysis, a sample preparation, and results. (CW)

  18. Determination of azide impurity in sartans using reversed-phase HPLC with UV detection.

    PubMed

    Gričar, Maja; Andrenšek, Samo

    2016-06-01

    A simple and robust, gradient HPLC method was developed for determination of azide ppm (μgg(-1)) levels in different sartans (irbesartan, candesartan, valsartan). The sartan was dissolved in 0.1M NaOH. Then pH was adjusted to 4.5 with 20% H3PO4 followed by dilution with water. Precipitated API was removed by filtration using 0.45μm membrane PVDF (Polyvinylidene Fluoride) membrane filter, and supernatant was analyzed by gradient elution HPLC at room temperature with Hydro RP HPLC 250×4.6mm, 4μm column and UV detection at 205nm. The best sensitivity was achieved by UV detection cell with 60mm optical path length: LOD 0.17μgg(-1) and LOQ 0.84μgg(-1) for azide. The USP requirement for maximum azide content in irbesartan is 10μgg(-1). The analytical method was validated as per International Conference on Harmonization (ICH) guidelines with respect to system precision, intraday precision (repeatability), intermediate precision (ruggedness), linearity, quantitation limit, detection limit, accuracy, standard and sample solution stability, robustness and selectivity/specificity. The method was linear in the range from LOQ (0.84μgg(-1)) to 101μgg(-1) of azide. The correlation coefficient was 0.9999 and bias on y-axis for 84μgg(-1) test concentration was 0.33%. The accuracy of the method was established based on the recovery obtained between 94.0% and 103.0% for azide. Standard and sample solutions were stable for at least 48h at room temperature and in refrigerator. The method was found to be robust for variation in column temperature (±5°C) and mobile phase flow rate (±0.2mLmin(-1)) and selective for anions such as bromide, nitrate, nitrite, formate and acetate. PMID:26999319

  19. New HPLC evidence on endogenous tauret in retina and pigment epithelium.

    PubMed

    Petrosian, A M; Haroutounian, J E; Gundersen, T E; Blomhoff, R; Fugelli, K; Kanli, H

    2000-01-01

    This investigation was improve the separation for tauret (retinylidene taurine) and to compare its content in the retina under dark and light adaptation. To prevent tauret hydrolysis, retinal samples were quickly frozen and lyophilized. Methanol extracts of dried retina and pigment epithelium from both dark- or light-adapted frogs, Rana ridibunda, were injected onto HPLC. Synthetic standard tauret appeared at 4.7 min after the solvent front. At the same time, an endogenous substance was eluted from the mixed retinal and pigment epithelial samples. The UV spectra of this endogenous compound matched with the spectra of synthetic tauret obtained under identical conditions, with lambda(max) = 446 nm at peak. We conclude that the HPLC system used permitted full separation of tauret from the methanol extracts of the retina and pigment epithelium. TLC and further HPLC analysis have shown that tauret quantities were several times higher in the retina and pigment epithelium of the frogs adapted to dark compared with those light-adapted (about 4 h under 1000 1x illumination). Tauret based vitamin A transport is probably involved in other systems as well, where along with its other known beneficial effects taurine probably is necessary to facilitate vitamin A transport.

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

    USGS Publications Warehouse

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

    2008-01-01

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

  1. Chromatographic purification and characterization of B-phycoerythrin from Porphyridium cruentum. Semipreparative high-performance liquid chromatographic separation and characterization of its subunits.

    PubMed

    Bermejo, R; Talavera, E M; Alvarez-Pez, J M

    2001-05-11

    A fast preparative two-step chromatographic method for purification of B-phycoerythrin from Porphyridium cruentum is described. This biliprotein was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielding three closely migrating bands corresponding to its three subunits. Baseline separation of its alpha-, beta- and gamma-subunits was achieved by a reversed-phase HPLC gradient semipreparative method with a C4 large-pore column and a solvent system consisting of 0.05% trifluoroacetic acid (TFA) in water and 0.05% TFA in acetonitrile. B-Phycoerythrin in different aggregation states and its subunits have been spectroscopically characterized. Hexameric B-phycoerythrin has similar secondary and tertiary structure than dissociated B-phycoerythrin determined by circular dichroism.

  2. Development of a validated RP-LC/ESI-MS-MS method for separation, identification and determination of related substances of tamsulosin in bulk drugs and formulations.

    PubMed

    Nageswara Rao, R; Kumar Talluri, M V N; Narasa Raju, A; Shinde, Dhananjay D; Ramanjaneyulu, G S

    2008-01-01

    A reversed-phase high performance liquid chromatographic (RP-HPLC) method for evaluation of purity of tamsulosin in bulk drugs and pharmaceuticals was developed. The separation was accomplished on an Inertsil C(18) column using 10 mM ammonium acetate: acetonitrile as a mobile phase in a gradient elution mode. A photodiode array detector set at 280 nm was used for detection. The impurities were identified by ESI-MS-MS. The detection limits were 0.06-0.11 microg/ml. The method was validated with respect to accuracy, precision, linearity, ruggedness and limits of detection and quantification. It finds application not only for monitoring the reactions during the process development but also on quality assurance of tamsulosin.

  3. Gas separating

    DOEpatents

    Gollan, Arye

    1988-01-01

    Feed gas is directed tangentially along the non-skin surface of gas separation membrane modules comprising a cylindrical bundle of parallel contiguous hollow fibers supported to allow feed gas to flow from an inlet at one end of a cylindrical housing through the bores of the bundled fibers to an outlet at the other end while a component of the feed gas permeates through the fibers, each having the skin side on the outside, through a permeate outlet in the cylindrical casing.

  4. Gas separating

    DOEpatents

    Gollan, Arye Z. [Newton, MA

    1990-12-25

    Feed gas is directed tangentially along the non-skin surface of gas separation membrane modules comprising a cylindrical bundle of parallel contiguous hollow fibers supported to allow feed gas to flow from an inlet at one end of a cylindrical housing through the bores of the bundled fibers to an outlet at the other end while a component of the feed gas permeates through the fibers, each having the skin side on the outside, through a permeate outlet in the cylindrical casing.

  5. Separation system

    DOEpatents

    Rubin, Leslie S.

    1986-01-01

    A separation system for dewatering radioactive waste materials includes a disposal container, drive structure for receiving the container, and means for releasably attaching the container to the drive structure. Separation structure disposed in the container adjacent the inner surface of the side wall structure retains solids while allowing passage of liquids. Inlet port structure in the container top wall is normally closed by first valve structure that is centrifugally actuated to open the inlet port and discharge port structure at the container periphery receives liquid that passes through the separation structure and is normally closed by second valve structure that is centrifugally actuated to open the discharge ports. The container also includes coupling structure for releasable engagement with the centrifugal drive structure. Centrifugal force produced when the container is driven in rotation by the drive structure opens the valve structures, and radioactive waste material introduced into the container through the open inlet port is dewatered, and the waste is compacted. The ports are automatically closed by the valves when the container drum is not subjected to centrifugal force such that containment effectiveness is enhanced and exposure of personnel to radioactive materials is minimized.

  6. Component separations.

    PubMed

    Heller, Lior; McNichols, Colton H; Ramirez, Oscar M

    2012-02-01

    Component separation is a technique used to provide adequate coverage for midline abdominal wall defects such as a large ventral hernia. This surgical technique is based on subcutaneous lateral dissection, fasciotomy lateral to the rectus abdominis muscle, and dissection on the plane between external and internal oblique muscles with medial advancement of the block that includes the rectus muscle and its fascia. This release allows for medial advancement of the fascia and closure of up to 20-cm wide defects in the midline area. Since its original description, components separation technique underwent multiple modifications with the ultimate goal to decrease the morbidity associated with the traditional procedure. The extensive subcutaneous lateral dissection had been associated with ischemia of the midline skin edges, wound dehiscence, infection, and seroma. Although the current trend is to proceed with minimally invasive component separation and to reinforce the fascia with mesh, the basic principles of the techniques as described by Ramirez et al in 1990 have not changed over the years. Surgeons who deal with the management of abdominal wall defects are highly encouraged to include this technique in their collection of treatment options.

  7. Gradient boosting machines, a tutorial

    PubMed Central

    Natekin, Alexey; Knoll, Alois

    2013-01-01

    Gradient boosting machines are a family of powerful machine-learning techniques that have shown considerable success in a wide range of practical applications. They are highly customizable to the particular needs of the application, like being learned with respect to different loss functions. This article gives a tutorial introduction into the methodology of gradient boosting methods with a strong focus on machine learning aspects of modeling. A theoretical information is complemented with descriptive examples and illustrations which cover all the stages of the gradient boosting model design. Considerations on handling the model complexity are discussed. Three practical examples of gradient boosting applications are presented and comprehensively analyzed. PMID:24409142

  8. The limits of the separation power of unidimensional column liquid chromatography.

    PubMed

    Guiochon, Georges

    2006-09-01

    The practical limit of the separation power of HPLC depends on time, money, and skill. That is it depends on the time available for the analysis, on the quality and performance of the pump and hardware and particularly on the maximum pressure at which the pump can deliver the mobile phase to the column, and on the temperature at which the column can be operated. It also depends on the properties of the packing material selected (e.g., its particle size, its pore geometry, and its connectivity) and on the packing method used since it affects the coefficients of the HETP equation. Finally, it depends on the thermal stability of the sample and the packing material. The complexity of the sample also plays an important role in that it determines whether the analysis should be made under isocratic, isothermal conditions, in gradient elution, in temperature programming, or with a combination of both types of programming. The various phenomena that affect column properties and separation performance are discussed. Past achievements suggest that columns providing efficiencies in excess of a million plates in less than 1 day are within the grasp of current technology. The possibility of further advances are considered.

  9. Development and evaluation of a new method for the determination of the carotenoid content in selected vegetables by HPLC and HPLC-MS-MS.

    PubMed

    Huck, C W; Popp, M; Scherz, H; Bonn, G K

    2000-10-01

    Epidemologic studies have shown inverse correlation between the consumption of carotenoid-rich vegetables and the incidence of cancer. Therefore, analytical techniques for the quantitative determination of carotenoids in complex sample matrices are important. The most used method is reversed-phase (RP)-high-performance liquid chromatography (HPLC). In this study, seventeen mobile-phase systems described in the literature and six RP-HPLC columns with differences in particle size and porosity are evaluated. Derived from these results, a new mobile-phase (acetonitrile, methanol, chloroform, and n-heptane) including solvent modifiers is presented, which allows an improved and more efficient separation of carotenoids. From all columns tested, the best chromatographic parameters are found using a silica C18 column (250 x 2 mm, 5 microm, 100 A). As was found, absorbance detection at 450 nm allows the determination of the carotenoids down to the picogram range with good linearity (R2 > 0.98). For the identification and quantitation of carotenoids in complex sample matrices (containing additionally other ultraviolet-absorbing compounds), the optimized RP chromatographic system is coupled to a mass spectrometer (MS) using an atmospheric pressure ionization interface. The calibration plots show high linearity (R2 > 0.99), and the detection limit is found in the lower nanogram range. Furthermore, collision-induced dissociation in the ion source allows for the identification of carotenoids by their characteristic fragmentation pathways. In this study, a total of nine species of vegetables commonly consumed in Central Europe are analyzed for their contents of carotenoids (namely lutein, zeaxanthin, beta-cryptoxanthin, and beta-carotene) by RP-HPLC and RP-HPLC-MS-MS. It is found that good sources for lutein are spinach, kale, and broccoli, and sources for beta-carotene are broccoli, spinach, kale, carrots, and tomatoes. This new method is an improvement for the identification and

  10. Simultaneous analysis of neuroendocrine tumor markers by HPLC-electrochemical detection.

    PubMed

    Manickum, T

    2009-12-15

    A validated, high pressure liquid chromatographic (HPLC) method for simultaneous quantitation of urinary catecholic acids 4-hydroxy-3-methoxymandelic acid (HMMA) (vanylmandelic acid) (VMA), 4-hydroxy-3-methoxyphenylacetic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) was developed. Sample preparation involved liquid-liquid extraction of acidified urine, containing iso-HMMA (IS) as internal standard, with ether, evaporation of the organic extract, followed by reconstitution of the residue in phosphate buffer at pH 3.3. After reversed-phase HPLC at 35 degrees C and separation on a Licrospher 125mmx4mm C(18) column (5microm particle size) with phosphate buffer (pH 3.5)-acetone (950:50, v/v) as eluent, quantitation is achieved by electrochemical detection using coulometric detection at a potential of +350mV. The method was successfully applied to routine diagnosis of neuroblastoma, carcinoid syndrome and pheochromocytoma. PMID:19926540

  11. [Preparation of ferulic acid, senkyunolide I and senkyunolide H from Ligusticum chuanxiong by preparative HPLC].

    PubMed

    Xiong, Yao-Kun; Liang, Shuang; Hong, Yan-Long; Yang, Xiu-Juan; Shen, Lan; Du, Yan; Feng, Yi

    2013-06-01

    Preparative HPLC was used to prepare ferulic acid, senkyunolide I and senkyunolide H from Ligusticum chuanxiong. The separation was conducted on a Shim-Pack Prep-ODS (20.0 mm x 250 mm, 5 microm) column with the mobile phase of methanol-0.2% glacial acetic acid (50:50)at the flow rate of 5 mL x min(-1). The detection wavelength was 278 nm, and the purity of each compound was detected by HPLC analysis. Spectral data analyses including UV, ESI-MS and NMR were used to identify their structures. This method is simple, fast, which is suitable for preparation of standard reference of ferulic acid, senkyunolide I and senkyunolide H from L. chuanxiong and can meet the requirement of new drug research and development. PMID:24066590

  12. A Rapid, Robust and Ultra-Sensitive HPLC Enantioseparation of β-Amino Alcohols.

    PubMed

    Dubey, Rituraj; Bhushan, Ravi

    2015-09-01

    Enantioseparation of amino acid analogues racemic β-amino alcohols has hitherto been thought impossible without prior derivatization. Method developers of chromatographic enantioseparation often face detection challenge due to low ultraviolet (UV) absorbance of these molecules. A new chiral derivatizing reagent, benzimidazole-(S)-naproxen amide, was synthesized to provide UV detectable chiral moiety for detection of respective diastereomers of eight β-amino alcohols (dl-alaninol, dl-leucinol, dl-prolinol, dl-phenylalaninol, dl-phenylglycinol, dl-valinol, dl-homophenylalaninol and dl-methioninol). An HPLC method has been developed and validated and both the diastereomers were separated within 31 min runtime. Excellent resolution to a pmol detection limit level was achieved. The developed HPLC method for enantioseparation of β-amino alcohols is the first-ever report of its own kind having such a high detection sensitivity. PMID:25824571

  13. The effect of re-dissolution solvents and HPLC columns on the analysis of mycosporine-like amino acids in the eulittoral macroalgae Prasiola crispa and Porphyra umbilicalis

    NASA Astrophysics Data System (ADS)

    Karsten, Ulf; Escoubeyrou, Karine; Charles, François

    2009-09-01

    Many macroalgal species that are regularly exposed to high solar radiation such as the eulittoral green alga Prasiola crispa and the red alga Porphyra umbilicalis synthesize and accumulate high concentrations of mycosporine-like amino acids (MAAs) as UV-sunscreen compounds. These substances are typically extracted with a widely used standard protocol following quantification by various high performance liquid chromatography (HPLC) techniques. However, further preparation steps prior to HPLC analysis as well as different HPLC column types have not been systematically checked regarding separation quality and reproducibility. Therefore pure methanol, distilled water and HPLC eluent were evaluated as re-dissolution solvent for dried Prasiola and Porphyra extracts, which were subsequently analyzed on three reversed-phase C8 and C18 HPLC columns. The data indicate that distilled water and the HPLC eluent gave almost identical peak patterns and MAA contents on the C8 and C18 columns. In contrast, the application of the widely used methanol led to double peaks or even the loss of specific peaks as well as to a strong decline in total MAA amounts ranging from about 35% of the maximum in P. crispa to 80% of the maximum in P. umbilicalis. Consequently, methanol should be avoided as re-dissolution solvent for the HPLC sample preparation. An improved protocol for the MAA analysis in macroalgae in combination with a reliable C18 column is suggested.

  14. Flow separation of currents in shallow water

    USGS Publications Warehouse

    Signell, Richard P.

    1989-01-01

    Flow separation of currents in shallow coastal areas is investigated using a boundary layer model for two-dimensional (depth-averaged) tidal flow past an elliptic headland. If the shoaling region near the coast is narrow compared to the scale of the headland, bottom friction causes the flow to separate just downstream of the point where the pressure gradient switches from favoring to adverse. As long as the shoaling region at the coast is well resolved, the inclusion of eddy viscosity and a no-slip boundary condition have no effect on this result. An approximate analytic solution for the pressure gradient along the boundary is obtained by assuming the flow away from the immediate vicinity of the boundary is irrotational. On the basis of the pressure gradient obtained from the irrotational flow solution, flow separation is a strong function of the headland aspect ratio, an equivalent Reynolds number, and a Keulegan-Carpenter number.

  15. Amine-phenyl multi-component gradient stationary phases.

    PubMed

    Dewoolkar, Veeren C; Kannan, Balamurali; Ashraf, Kayesh M; Higgins, Daniel A; Collinson, Maryanne M

    2015-09-01

    Continuous multi-component gradients in amine and phenyl groups were fabricated using controlled rate infusion (CRI). Solutions prepared from either 3-aminopropyltriethoxysilane (APTEOS) or phenyltrimethoxysilane (PTMOS) were infused, in a sequential fashion, at a controlled rate into an empty graduated cylinder housing a vertically aligned thin layer chromatography (TLC) plate. The hydrolyzed precursors reacted with an abundance of silanol (SiOH) groups on the TLC plates, covalently attaching the functionalized silane to its surface. The extent of modification by phenyl and amine was determined by the kinetics of each reaction and the exposure time at each point along the TLC plate. The local concentrations of phenyl and amine were measured using diffuse reflectance spectroscopy and X-ray photoelectron spectroscopy, respectively. The profile of the multi-component gradients strongly depended on the order of infusion, the direction of the gradient and the presence of available surface silanol groups. A slightly higher amount of phenyl can be deposited on the TLC plate by first modifying its surface with amine groups as they serve as a catalyst, enhancing condensation. Separation of water- and fat-soluble vitamins and the control of retention factors were demonstrated on the multi-component gradient TLC plates. Uniformly modified and single-component TLC plates gave different separations compared to the multi-component gradient plates. The retention factors of the individual vitamins depended on the order of surface modification, the spotting end, and whether the multi-component gradients align or oppose each other. PMID:26255112

  16. Separation of cells from the rat anterior pituitary gland

    NASA Technical Reports Server (NTRS)

    Hymer, Wesley C.; Hatfield, J. Michael

    1983-01-01

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

  17. Gas separating

    DOEpatents

    Gollan, A.

    1988-03-29

    Feed gas is directed tangentially along the non-skin surface of gas separation membrane modules comprising a cylindrical bundle of parallel contiguous hollow fibers supported to allow feed gas to flow from an inlet at one end of a cylindrical housing through the bores of the bundled fibers to an outlet at the other end while a component of the feed gas permeates through the fibers, each having the skin side on the outside, through a permeate outlet in the cylindrical casing. 3 figs.

  18. Gas separating

    DOEpatents

    Gollan, A.Z.

    1990-12-25

    Feed gas is directed tangentially along the non-skin surface of gas separation membrane modules comprising a cylindrical bundle of parallel contiguous hollow fibers supported to allow feed gas to flow from an inlet at one end of a cylindrical housing through the bores of the bundled fibers to an outlet at the other end while a component of the feed gas permeates through the fibers, each having the skin side on the outside, through a permeate outlet in the cylindrical casing. 3 figs.

  19. Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay.

    PubMed

    Pais de Barros, Jean-Paul; Gautier, Thomas; Sali, Wahib; Adrie, Christophe; Choubley, Hélène; Charron, Emilie; Lalande, Caroline; Le Guern, Naig; Deckert, Valérie; Monchi, Mehran; Quenot, Jean-Pierre; Lagrost, Laurent

    2015-07-01

    Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo. PMID:26023073

  20. Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[S

    PubMed Central

    Pais de Barros, Jean-Paul; Gautier, Thomas; Sali, Wahib; Adrie, Christophe; Choubley, Hélène; Charron, Emilie; Lalande, Caroline; Le Guern, Naig; Deckert, Valérie; Monchi, Mehran; Quenot, Jean-Pierre; Lagrost, Laurent

    2015-01-01

    Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo. PMID:26023073

  1. Separation of carbon nanotubes into chirally enriched fractions

    DOEpatents

    Doorn, Stephen K.; Niyogi, Sandip

    2012-04-10

    A mixture of single-walled carbon nanotubes ("SWNTs") is separated into fractions of enriched chirality by preparing an aqueous suspension of a mixture of SWNTs and a surfactant, injecting a portion of the suspension on a column of separation medium having a density gradient, and centrifuging the column. In some embodiments, salt is added prior to centrifugation. In other embodiments, the centrifugation is performed at a temperature below room temperature. Fractions separate as colored bands in the column. The diameter of the separated SWNTs decreases with increasing density along the gradient of the column. The colored bands can be withdrawn separately from the column.

  2. Density Gradients in Chemistry Teaching

    ERIC Educational Resources Information Center

    Miller, P. J.

    1972-01-01

    Outlines experiments in which a density gradient might be used to advantage. A density gradient consists of a column of liquid, the composition and density of which varies along its length. The procedure can be used in analysis of solutions and mixtures and in density measures of solids. (Author/TS)

  3. Empirical equation estimates geothermal gradients

    SciTech Connect

    Kutasov, I.M. )

    1995-01-02

    An empirical equation can estimate geothermal (natural) temperature profiles in new exploration areas. These gradients are useful for cement slurry and mud design and for improving electrical and temperature log interpretation. Downhole circulating temperature logs and surface outlet temperatures are used for predicting the geothermal gradients.

  4. Multilayer High-Gradient Insulators

    SciTech Connect

    Harris, J R

    2006-08-16

    Multilayer High-Gradient Insulators are vacuum insulating structures composed of thin, alternating layers of dielectric and metal. They are currently being developed for application to high-current accelerators and related pulsed power systems. This paper describes some of the High-Gradient Insulator research currently being conducted at Lawrence Livermore National Laboratory.

  5. Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC-ESI/MS.

    PubMed

    Liu, Shiming; Chen, Kaoshan; Schliemann, Willibald; Strack, Dieter

    2005-01-01

    A simple method involving polyamide column chromatography in combination with HPLC-PAD and HPLC-ESI/MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4 degrees C from which two main compounds were purified by semi-preparative HPLC. In comparison with the UV and ESI-MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction/separation technique was validated using an internal standard method.

  6. Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance.

    PubMed

    Gfrörer, P; Schewitz, J; Pusecker, K; Tseng, L H; Albert, K; Bayer, E

    1999-01-01

    Coupling of gradient capillary electrochromatography (gradient CEC) and capillary zone electrophoresis (CZE) with nuclear magnetic resonance spectroscopy (NMR) was performed using a recently developed capillary NMR interface. This technique was applied for the analysis of pharmaceuticals and food. An analgesic was investigated using isocratic and gradient continuous-flow CEC-NMR. Comparison of the results demonstrated the superiority of gradient CEC over isocratic CEC. Aspartame and caffeine, both ingredients of soft beverages, were separated and analyzed by continuous flow CZE-NMR. The order of elution could be reversed by altering the pH. This reversal led to an increased sample concentration in the NMR detection cell, thus allowing the acquisition of a totally correlated spectroscopy (TOCSY) two-dimensional (2-D) spectrum of the synthetic peptide aspartame. PMID:10065951

  7. Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance.

    PubMed

    Gfrörer, P; Schewitz, J; Pusecker, K; Tseng, L H; Albert, K; Bayer, E

    1999-01-01

    Coupling of gradient capillary electrochromatography (gradient CEC) and capillary zone electrophoresis (CZE) with nuclear magnetic resonance spectroscopy (NMR) was performed using a recently developed capillary NMR interface. This technique was applied for the analysis of pharmaceuticals and food. An analgesic was investigated using isocratic and gradient continuous-flow CEC-NMR. Comparison of the results demonstrated the superiority of gradient CEC over isocratic CEC. Aspartame and caffeine, both ingredients of soft beverages, were separated and analyzed by continuous flow CZE-NMR. The order of elution could be reversed by altering the pH. This reversal led to an increased sample concentration in the NMR detection cell, thus allowing the acquisition of a totally correlated spectroscopy (TOCSY) two-dimensional (2-D) spectrum of the synthetic peptide aspartame.

  8. Chemical characterization of lipids in speleothems and cave drip water by organic trace analysis (HPLC-ESI-IT/MS) and its potential for the investigation of palaeoenvironmental changes

    NASA Astrophysics Data System (ADS)

    Bosle, J.; Scholz, D.; Hoffmann, T.

    2012-04-01

    Cave drip water, speleothems and the proxies preserved within them have significant potential to record palaeoenvironmental changes in the regional vegetation [1]. The use of stalagmites provides valuable information because they form a chemically closed system which does not change much after lithification, they grow continuously and are amenable to precise Th/U-dating [2]. The most common proxies measured in speleothems are inorganic proxies, in particular oxygen isotopes, but more recently the importance of organic matter analyses in this field is examined. This study focuses on the research of lipid biomarkers. The lipids contained in stalagmites originate from the overlying soil and different plants, bacteria and fungi. Therefore different compositions of lipids may provide records of environmental changes [3]. In the following the development of a new method for the extraction of saturated free fatty acids from cave drip water and their measurement by RP-HPLC-ESI-IT/MS (reversed phase high performance liquid chromatography coupled to electrospray ion trap mass spectrometry) is presented. Four different fatty acids (myristic acid, palmitic acid, stearic acid and arachidic acid) with chain lengths from C14 to C20 were applied as analytical standards. A mixture of these was used to optimize the separation by HPLC. The analytes were measured in MRM mode (multiple reactions monitoring) and negative polarity (m/z: 227, 255, 283, 311) therefore a time consuming derivatisation of the fatty acids was not necessary. By using a certain gradient program an adequate separation of the standards was accomplished. Likewise the realization of calibration curves of the different fatty acids showed that quantitative analyses are possible as well. To simulate the extraction of the analytes from cave drip water a spiking experiment with the analytical standards was performed. Three water samples were spiked with the same amount of the fatty acids to test the reproducibility of

  9. Density-Gradient Determination of Osmotic Potential in Plant Cells

    ERIC Educational Resources Information Center

    Nabors, Murray W.

    1973-01-01

    Describes a method for measuring osmotic potential which is suitable for high school and college biology classes. This method introduces students to the hard-to-visualize technique of using density gradients to separate cells or cell constituents of differing densities. (JR)

  10. Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Phenoxyethanol, Methylparaben, Propylparaben, Mometasone Furoate, and Tazarotene in Topical Pharmaceutical Dosage Formulation

    PubMed Central

    Roy, Chinmoy; Chakrabarty, Jitamanyu

    2013-01-01

    A stability-indicating RP-HPLC method has been developed and validated for the simultaneous determination of phenoxyethanol (PE), methylparaben (MP), propylparaben (PP), mometasone furoate (MF), and tazarotene (TA) in topical pharmaceutical dosage formulation. The desired chromatographic separation was achieved on the Waters X-Bridge™ C18 (50×4.6mm, 3.5μ) column using gradient elution at 256 nm detection wavelength. The optimized mobile phase consisted of 0.1%v/v orthophosphoric acid in water as solvent-A and acetonitrile as solvent-B. The method showed linearity over the range of 5.88–61.76 μg/mL, 0.18–62.36 μg/mL, 0.17–6.26 μg/mL, 0.47–31.22 μg/mL, and 0.44–30.45 μg/mL for PE, MP, PP, MF, and TA, respectively. The recovery for all of the components was in the range of 98–102%. The stability-indicating capability of the developed method was established by analysing the forced degradation samples, in which the spectral purity of PE, MP, PP, MF, and TA along with the separation of degradation products from the analyte peaks was achieved. The proposed method was successfully applied for the quantitative determination of PE, MP, PP, MF, and TA in a cream sample. PMID:24482766

  11. Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Phenoxyethanol, Methylparaben, Propylparaben, Mometasone Furoate, and Tazarotene in Topical Pharmaceutical Dosage Formulation.

    PubMed

    Roy, Chinmoy; Chakrabarty, Jitamanyu

    2013-12-01

    A stability-indicating RP-HPLC method has been developed and validated for the simultaneous determination of phenoxyethanol (PE), methylparaben (MP), propylparaben (PP), mometasone furoate (MF), and tazarotene (TA) in topical pharmaceutical dosage formulation. The desired chromatographic separation was achieved on the Waters X-Bridge™ C18 (50×4.6mm, 3.5μ) column using gradient elution at 256 nm detection wavelength. The optimized mobile phase consisted of 0.1%v/v orthophosphoric acid in water as solvent-A and acetonitrile as solvent-B. The method showed linearity over the range of 5.88-61.76 μg/mL, 0.18-62.36 μg/mL, 0.17-6.26 μg/mL, 0.47-31.22 μg/mL, and 0.44-30.45 μg/mL for PE, MP, PP, MF, and TA, respectively. The recovery for all of the components was in the range of 98-102%. The stability-indicating capability of the developed method was established by analysing the forced degradation samples, in which the spectral purity of PE, MP, PP, MF, and TA along with the separation of degradation products from the analyte peaks was achieved. The proposed method was successfully applied for the quantitative determination of PE, MP, PP, MF, and TA in a cream sample.

  12. Stochastic approach for an unbiased estimation of the probability of a successful separation in conventional chromatography and sequential elution liquid chromatography.

    PubMed

    Ennis, Erin J; Foley, Joe P

    2016-07-15

    A stochastic approach was utilized to estimate the probability of a successful isocratic or gradient separation in conventional chromatography for numbers of sample components, peak capacities, and saturation factors ranging from 2 to 30, 20-300, and 0.017-1, respectively. The stochastic probabilities were obtained under conditions of (i) constant peak width ("gradient" conditions) and (ii) peak width increasing linearly with time ("isocratic/constant N" conditions). The isocratic and gradient probabilities obtained stochastically were compared with the probabilities predicted by Martin et al. [Anal. Chem., 58 (1986) 2200-2207] and Davis and Stoll [J. Chromatogr. A, (2014) 128-142]; for a given number of components and peak capacity the same trend is always observed: probability obtained with the isocratic stochastic approachgradient stochastic approach≤probability predicted by Davis and Stoll < probability predicted by Martin et al. The differences are explained by the positive bias of the Martin equation and the lower average resolution observed for the isocratic simulations compared to the gradient simulations with the same peak capacity. When the stochastic results are applied to conventional HPLC and sequential elution liquid chromatography (SE-LC), the latter is shown to provide much greater probabilities of success for moderately complex samples (e.g., PHPLC=31.2% versus PSE-LC=69.1% for 12 components and the same analysis time). For a given number of components, the density of probability data provided over the range of peak capacities is sufficient to allow accurate interpolation of probabilities for peak capacities not reported, <1.5% error for saturation factors <0.20. Additional applications for the stochastic approach include isothermal and programmed-temperature gas chromatography.

  13. Gradient zone boundary control in salt gradient solar ponds

    DOEpatents

    Hull, John R.

    1984-01-01

    A method and apparatus for suppressing zone boundary migration in a salt gradient solar pond includes extending perforated membranes across the pond at the boundaries, between the convective and non-convective zones, the perforations being small enough in size to prevent individual turbulence disturbances from penetrating the hole, but being large enough to allow easy molecular diffusion of salt thereby preventing the formation of convective zones in the gradient layer. The total area of the perforations is a sizable fraction of the membrane area to allow sufficient salt diffusion while preventing turbulent entrainment into the gradient zone.

  14. Hand-Portable Gradient Capillary Liquid Chromatography Pumping System.

    PubMed

    Sharma, Sonika; Plistil, Alex; Barnett, Hal E; Tolley, H Dennis; Farnsworth, Paul B; Stearns, Stanley D; Lee, Milton L

    2015-10-20

    In this work, a novel splitless nanoflow gradient generator integrated with a stop-flow injector was developed and evaluated using an on-column UV-absorption detector. The gradient pumping system consisted of two nanoflow pumps controlled by micro stepper motors, a mixer connected to a serpentine tube, and a high-pressure valve. The gradient system weighed only 4 kg (9 lbs) and could generate up to 55 MPa (8000 psi) pressure. The system could operate using a 24 V DC battery and required 1.2 A for operation. The total volume capacity of the pump was 74 μL, and a sample volume of 60 nL could be injected. The system provided accurate nanoflow rates as low as 10 nL/min without employing a splitter, making it ideal for capillary column use. The gradient dwell volume was calculated to be 1.3 μL, which created a delay of approximately 4 min with a typical flow rate of 350 nL/min. Gradient performance was evaluated for gradient step accuracy, and excellent reproducibility was obtained in day-to-day experiments (RSD < 1.2%, n = 4). Linear gradient reproducibility was tested by separating a three-component pesticide mixture on a poly(ethylene glycol) diacrylate (PEGDA) monolithic column. The retention time reproducibility was very good in run-to-run experiments (RSD < 1.42%, n = 4). Finally, excellent separation of five phenols was demonstrated using the nanoflow gradient system. PMID:26378495

  15. Particle separation

    NASA Technical Reports Server (NTRS)

    Moosmuller, Hans (Inventor); Chakrabarty, Rajan K. (Inventor); Arnott, W. Patrick (Inventor)

    2011-01-01

    Embodiments of a method for selecting particles, such as based on their morphology, is disclosed. In a particular example, the particles are charged and acquire different amounts of charge, or have different charge distributions, based on their morphology. The particles are then sorted based on their flow properties. In a specific example, the particles are sorted using a differential mobility analyzer, which sorts particles, at least in part, based on their electrical mobility. Given a population of particles with similar electrical mobilities, the disclosed process can be used to sort particles based on the net charge carried by the particle, and thus, given the relationship between charge and morphology, separate the particles based on their morphology.

  16. Particle separation

    DOEpatents

    Moosmuller, Hans; Chakrabarty, Rajan K.; Arnott, W. Patrick

    2011-04-26

    Embodiments of a method for selecting particles, such as based on their morphology, is disclosed. In a particular example, the particles are charged and acquire different amounts of charge, or have different charge distributions, based on their morphology. The particles are then sorted based on their flow properties. In a specific example, the particles are sorted using a differential mobility analyzer, which sorts particles, at least in part, based on their electrical mobility. Given a population of particles with similar electrical mobilities, the disclosed process can be used to sort particles based on the net charge carried by the particle, and thus, given the relationship between charge and morphology, separate the particles based on their morphology.

  17. High field gradient particle accelerator

    DOEpatents

    Nation, J.A.; Greenwald, S.

    1989-05-30

    A high electric field gradient electron accelerator utilizing short duration, microwave radiation, and capable of operating at high field gradients for high energy physics applications or at reduced electric field gradients for high average current intermediate energy accelerator applications is disclosed. Particles are accelerated in a smooth bore, periodic undulating waveguide, wherein the period is so selected that the particles slip an integral number of cycles of the r.f. wave every period of the structure. This phase step of the particles produces substantially continuous acceleration in a traveling wave without transverse magnetic or other guide means for the particle. 10 figs.

  18. High field gradient particle accelerator

    DOEpatents

    Nation, John A.; Greenwald, Shlomo

    1989-01-01

    A high electric field gradient electron accelerator utilizing short duration, microwave radiation, and capable of operating at high field gradients for high energy physics applications or at reduced electric field gradients for high average current intermediate energy accelerator applications. Particles are accelerated in a smooth bore, periodic undulating waveguide, wherein the period is so selected that the particles slip an integral number of cycles of the r.f. wave every period of the structure. This phase step of the particles produces substantially continuous acceleration in a traveling wave without transverse magnetic or other guide means for the particle.

  19. HPLC-DAD protein kinase inhibitor analysis in human serum.

    PubMed

    Dziadosz, Marek; Lessig, Rüdiger; Bartels, Heidemarie

    2012-04-15

    We here describe an HPLC-DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib - internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250 mm × 4 mm column maintained at 30 ± 1 °C, and with a mobile phase of 0.05 M H(3)PO(4)/KH(2)PO(4) (pH=2.3)-acetonitrile (7:3, v/v) at a flow rate of 0.7 mL/min. A simple and fast sample preparation sequence with liquid-liquid extraction led to good recoveries (73-90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20-10,000 ng/mL) and an LOQ of 20 ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range. PMID:22425385

  20. The rapid isolation of highly purified cadmium metallothioneins using HPLC

    SciTech Connect

    Vella, G.; Bylander, J.; Hazen-martin, D.; O'Connor, K.; Phoebe, C. Millipore Corp., Milford, MA )

    1991-03-11

    The central role of the proximal tubule in the renal toxicity due to cadmium (Cd) poisoning has been well documented, however, the mechanisms leading to the onset of this nephrotoxic effect are presently not well understood. The most likely mechanism for the initiation of toxicity involves the accumulation of Cd in the liver where it induces the formation of, and is complexed with the metallothioneins (MT-I and MT-II) followed by release from the liver and subsequent absorption by the proximal tubule. Methods for obtaining MTs for specific RIA and immunohistochemistry have been hampered by difficulties in removing impurities. To obtain highly purified Cd-MTs for use in RIA as well as to assay the relative affects of exposure to Cd-MT and ionic Cd in vitro on human proximal tubule cells, Cd-MT-I and Cd-MT-II were prepared from rat liver for use in these studies using a rapid two step chromatographic procedure employing gel filtration and high performance anion exchange chromatography. Repeated HPLC separations revealed a time dependent conversion of a non-metallothionein protein component to form which coeluted with Cd-MT-I during the ion exchange separation. This was preventable by cooling the sample or by adding proteinase inhibitors. This method demonstrated the importance of speed in the purification which permitted the isolation of highly purified Cd-MTs in high yields. Due to the extreme sensitivity, this method may also be amenable as an alternative for a non-radioactive assay of Cd-MT formation and breakdown in toxicological studies.