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Sample records for interaction chromatography stationary

  1. Glucaminium ionic liquid-functionalized stationary phase for the separation of nucleosides in hydrophilic interaction chromatography.

    PubMed

    Jiang, Qiong; Zhang, Mingliang; Wang, Xusheng; Guo, Yong; Qiu, Hongdeng; Zhang, Shusheng

    2015-10-01

    A glucaminium-based ionic liquid stationary phase was prepared via facile epoxy-amine reaction and subsequent quaternization. Successful immobilization of glucaminium-based ionic liquid onto silica surface was validated by elemental analysis and infrared spectroscopy. The new stationary phase was evaluated for the separation of nucleosides in hydrophilic interaction liquid chromatography (HILIC). Effects of various factors, such as acetonitrile concentration, salt concentration, pH value, as well as column temperature, on the chromatographic behavior toward nucleosides were studied in detail. The results indicated that this new stationary phase can be used for separation of water-soluble polar substances in HILIC mode. The retention of solutes on the stationary phase was influenced by a mixed-mode retention mechanism with a combination of adsorptive and partitioning interactions.

  2. Effect of silica gel modification with cyclofructans on properties of hydrophilic interaction liquid chromatography stationary phases.

    PubMed

    Kozlík, Petr; Símová, Veronika; Kalíková, Květa; Bosáková, Zuzana; Armstrong, Daniel W; Tesařová, Eva

    2012-09-28

    Hydrophilic interaction liquid chromatography (HILIC) offers very good possibilities for separation of polar compounds as an alternative to reversed phase HPLC where polar compounds are not sufficiently retained. HILIC is becoming more popular for the analysis of biologically interesting (active) analytes. Various stationary phases are commercially available however, development of new materials (sorbents) suitable for HILIC systems still continues. Silica gel columns can be used directly but their modification can improve separation ability of the stationary phases. Cyclofructan-based stationary phases are demonstrated as possible HILIC columns in this work. The effect of silica gel modification by cyclofructan and a derivatized cyclofructan was studied in detail. HILIC separation systems with silica gel, cyclofructan and isopropyl cyclofructan modified silica stationary phases were compared. The detailed study of chromatographic behavior of peptides revealed that multimodal retention mechanism is present in systems with these stationary phases. Mobile phase composition changes the types of interactions and their strengths. It appears that ability to donate protons and dispersion forces are the main interactions that affect retention in HILIC with cyclofructan-based columns while they are less important in separation systems with bare silica stationary phase. Suitability of cyclofructan-based stationary phases in HILIC for separation of pentapeptides and nonapeptides was demonstrated.

  3. Glutathione-based zwitterionic stationary phase for hydrophilic interaction/cation-exchange mixed-mode chromatography.

    PubMed

    Shen, Aijin; Li, Xiuling; Dong, Xuefang; Wei, Jie; Guo, Zhimou; Liang, Xinmiao

    2013-11-01

    As a naturally hydrophilic peptide, glutathione was facilely immobilized onto silica surface to obtain a novel hydrophilic interaction/cation-exchange mixed-mode chromatographic stationary phase (Click TE-GSH) via copper-free "thiol-ene" click chemistry. The resulting material was characterized by solid state (13)C/CP MAS NMR and elemental analysis. The measurement of ζ-potential indicated the cation-exchange characteristics and adjustable surface charge density of Click TE-GSH material. The influence of acetonitrile content and pH value on the retention of ionic compounds was investigated for understanding the chromatographic behaviors. The results demonstrated that Click TE-GSH column could provide both hydrophilic and cation-exchange interaction. Taking advantage of the good hydrophilicity and inherent cation-exchange characteristics of Click TE-GSH material, the resolution of neutral fructosan with high degree of polymerization (DP), basic chitooligosaccharides and strongly acidic carrageenan oligosaccharides was successfully realized in hydrophilic interaction chromatography (HILIC), hydrophilic interaction/cation-exchange mixed-mode chromatography (HILIC/CEX), cation-exchange chromatography (CEX) and electrostatic repulsion/hydrophilic interaction chromatography (ERLIC). On the other hand, the separation of standard peptides varying in hydrophobicity/hydrophilicity and charge was achieved in both CEX and HILIC/CEX mode with high efficiency and distinct selectivity. To further demonstrate the versatility and applicability of Click TE-GSH stationary phase, the separation of a human serum albumin (HSA) tryptic digest was performed in HILIC/CEX mode. Peptides were adequately resolved and up to 86 HSA peptides were identified with sequence coverage of 85%. The results indicated the good potential of Click TE-GSH material in glycomics and proteomics.

  4. Design and evaluation of hydrolytically stable bidentate urea-type stationary phases for hydrophilic interaction chromatography.

    PubMed

    Kotoni, Dorina; D'Acquarica, Ilaria; Ciogli, Alessia; Villani, Claudio; Capitani, Donatella; Gasparrini, Francesco

    2012-04-06

    We have developed conceptually new stationary phases containing two bidentate urea-type functions suitable for the separation of a wide variety of polar compounds by hydrophilic interaction chromatography (HILIC) through a facile one-pot two-step procedure with the aim of obtaining high hydrolytic stability in a variety of elution conditions. The preparation of the new phases involves a first reaction of 1,2-ethylendiamine with (3-isocyanatopropyl)triethoxysilane to give an intermediate bis-urea with two pendant triethoxysilane functions, followed by anchoring on the silica surface. Two stationary phases were prepared, namely an urea-type stationary phase (USP-HILIC) and an urea-type phase bearing free amino groups (USP-HILIC-NH(2)), whereas silanization with 1,2-bis(trichlorosilyl)ethane yielded USP-HILIC-sil and USP-HILIC-NH(2)-sil phases, respectively. The silanization step aimed at forming a hydrophilic stable coating through cross-linking between adjacent silanols which prevents silica dissolution at alkaline pH. A full chemical characterization of the new materials has been obtained through solid-state NMR (both (29)Si and (13)C CPMAS) spectroscopy. A major application field of the bidentate urea-type stationary phase with free amino groups USP-HILIC-NH(2)-sil was sugars analysis, usually hampered by α/β anomer peak splitting and instability of the stationary phases under conditions normally employed to suppress it. Complex mixtures of mono-, di- and oligosaccharides were successfully resolved under mild chromatographic conditions, which also allowed an easy interface with mass spectrometry. The potential of such materials was shown in the separation of other highly polar compounds, including polyols, hydroxybenzoic acids, nucleobases, and vitamins.

  5. Tetraazacalix[2]arene[2]triazine modified silica gel: a novel multi-interaction stationary phase for mixed-mode chromatography.

    PubMed

    Zhao, Wenjie; Wang, Wenjing; Chang, Hong; Cui, Shiwei; Hu, Kai; He, Lijun; Lu, Kui; Liu, Jinxia; Wu, Yangjie; Qian, Jiang; Zhang, Shusheng

    2012-08-17

    A novel multi-interaction and mixed-mode stationary phase based on tetraazacalix[2]arene[2]triazine modified silica (NCS) was synthesized and characterized by infrared spectra, elemental analysis and thermogravimetric analysis. Mechanism involved in the chromatographic separation is the multi-interaction including hydrophobic, π-π, hydrogen-bonding, inclusion and anion-exchange interactions. Based on these interactions, successful separation could be achieved among polycyclic aromatic hydrocarbons, aromatic position isomers, organic bases and phenols in reversed-phase chromatography. Inorganic anions were also shown to be individually separated in anion-exchange chromatography by using the same column. Moreover, the results here also demonstrated that NCS based stationary phase could effectively reduce the adverse effect of residual silanol in the separation process. Such stationary phase with characteristics of multi-interaction mechanism and mixed-mode separation is potential for the analysis of complex samples.

  6. Imidazolium embedded C8 based stationary phase for simultaneous reversed-phase/hydrophilic interaction mixed-mode chromatography.

    PubMed

    Qiao, Xiaoqiang; Zhang, Lu; Zhang, Niu; Wang, Xin; Qin, Xinying; Yan, Hongyuan; Liu, Haiyan

    2015-06-26

    A new imidazolium embedded C8 based stationary phase (SIL-MPS-VOL) was facilely prepared by two steps and characterized by Fourier transform infrared spectrometry and thermogravimetric analysis. Due to the introduction of quaternary imidazolium group to the traditional C8 stationary phase, the developed SIL-MPS-VOL column demonstrated both reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) retention mechanisms. A series of hydrophobic and hydrophilic test samples, including benzene homologues, anilines, positional isomers, nucleosides and nucleotides, were used to evaluate the developed SIL-MPS-VOL stationary phase. A rapid separation time, high separation efficiency and planar selectivity were achieved, compared with the commercially available C8 column. Moreover, the developed stationary phase was further used to detect and separate of melamine in powdered infant formula and high polar component of secondary metabolites of Trichoderma, and improved separation efficiency was achieved, indicating the potential merits of the developed SIL-MPS-VOL stationary phase for simultaneous separation of complex hydrophobic and hydrophilic samples with high selectivity.

  7. Preparation and evaluation of 2-methylimidazolium-functionalized silica as a mixed-mode stationary phase for hydrophilic interaction and anion-exchange chromatography.

    PubMed

    Yang, Beibei; Liu, Houmei; Chen, Jia; Guan, Ming; Qiu, Hongdeng

    2016-10-14

    In this paper, a novel 2-methylimidazolium-functionalized silica stationary phase was prepared and further used for hydrophilic interaction and anion-exchange mixed-mode chromatography. The stationary phase was characterized by elemental analysis and Fourier transform infrared spectrometry. The chromatographic properties of this stationary phase were investigated by hydrophilic chromatography for the separation of nucleosides, nucleobases, water soluble vitamins, sulfonamides and saccharides, and ion chromatography for the separation of inorganic anions. The effect of acetonitrile content, salt concentration and pH values of the mobile phase on the retention of the stationary phases was also investigated. Compared with 1-methylimidazolium-functionalized silica stationary phase, this new stationary phase demonstrated similar or better separation selectivity. This new column demonstrated good performance and separation selectivity even better than a commercial hydrophilic column. Besides, 2-methylimidazolium-functionalized silica is possible to be modified again and used as a precursor to derivate some new stationary phases from the 3-position nitrogen.

  8. Description and Evaluation of Chiral Interactive Sites on Bonded Cyclodextrin Stationary Phases for Liquid Chromatography

    NASA Astrophysics Data System (ADS)

    Beesley, Thomas E.

    Development of chiral separations has been essential to the drug discovery and development process. The solubility requirements for a number of methods and/or the mobile phase requirements for application of certain detection systems have opened up many opportunities for cyclodextrin-based CSPs for liquid chromatography. Even though a few chiral stationary phases cover a wide area of enantioselectivity, they do not meet the entire needs of the industry. Cyclodextrin phases offer some unique mechanisms and opportunities to resolve chiral separation problems especially in the aqueous reversed-phase and non-aqueous polar organic modes. This chapter addresses the need to understand the chiral stationary phase structure, the mechanisms at work, and the role mobile phase composition plays in driving those mechanisms to produce enantioselectivity. In addition, the development of certain derivatives has played an essential part in expanding that basic role for certain chiral separations. What these derivatives contribute in concert with the basic structure is a critical part of the understanding to the effective use of these phases. During this study it was determined that the role of steric hindrance has been vastly underestimated, both to the extent that it has occurred and to its effectiveness for obtaining enantioselectivity. References to the entire 20-year history of the cyclodextrin phase development and application literature up to this current date have been reviewed and incorporated.

  9. Preparation of a novel carboxyl stationary phase by "thiol-ene" click chemistry for hydrophilic interaction chromatography.

    PubMed

    Peng, Xi-Tian; Liu, Tao; Ji, Shu-Xian; Feng, Yu-Qi

    2013-08-01

    A novel carboxyl-bonded silica stationary phase was prepared by "thiol-ene" click chemistry. The resultant Thiol-Click-COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol-Click-COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol-Click-COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol-Click-COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water-soluble vitamins was achieved.

  10. Synthesis, characterization, and application of a novel multifunctional stationary phase for hydrophilic interaction/reversed phase mixed-mode chromatography.

    PubMed

    Aral, Hayriye; Çelik, K Serdar; Altındağ, Ramazan; Aral, Tarık

    2017-11-01

    A novel multifunctional stationary phase based on silica gel was synthesised starting from L- isoleucine and 4-phenylbutylamine and evaluated as a hydrophilic interaction/reversed-phase mixed-mode stationary phase for high-performance liquid chromatography (HPLC). The prepared stationary phase was characterized by elemental analysis, infrared spectroscopy (IR), scanning electron microscopy (SEM), Brunauer, Emmett and Teller (BET) and solid-state (13)C nuclear magnetic resonance (NMR). The mechanisms involved in the chromatographic separation are multi-interaction, including hydrophobic, π-π, hydrogen-bonding, dipole-dipole and ion-dipole interactions. Based on these interactions, successful separation could be achieved among several aromatic compounds having different polarities under both hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) condition. Nucleotides/nucleosides were separated in the HILIC mode. The effects of different separation conditions, such as pH value, mobile-phase content, column temperature, buffer concentration and flow rate, on the separation of nucleotides/nucleosides in HILIC mode were investigated. The seven nucleotides/nucleosides were separated within 22min, while six of them were separated within 10min by isocratic elution. To determine the influence of the new multifunctional stationary phase under the RP condition, a number of moderately and weakly polar and nonpolar compounds, such as 10 substituted anilines and eight substituted phenols were separated successfully under the RP condition within 14 and 15min, respectively. Additionally, nine mixtures of polar/nonpolar test compounds were simultaneously separated within 19min, while seven of them were separated within 12min, under HILIC/RP mixed-mode conditions. Chromatographic parameters, such as the retention factor and peak asymmetry factor, were calculated for all of the analytes, while the theoretical plate number was calculated for analytes separated

  11. Preparation of a weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography stationary phase for protein separation using click chemistry.

    PubMed

    Zhao, Kailou; Yang, Fan; Xia, Hongjun; Wang, Fei; Song, Qingguo; Bai, Quan

    2015-03-01

    In this study, 3-diethylamino-1-propyne was covalently bonded to the azide-silica by a click reaction to obtain a novel dual-function mixed-mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high-salt-concentration mobile phase and weak anion exchange character in a low-salt-concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed-mode chromatography stationary phase, a new off-line two-dimensional liquid chromatography technology using only a single dual-function mixed-mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.

  12. Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation.

    PubMed

    Zhao, Kailou; Yang, Li; Wang, Xuejiao; Bai, Quan; Yang, Fan; Wang, Fei

    2012-08-30

    We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid

  13. Preparation, characterization and application of a reversed phase liquid chromatography/hydrophilic interaction chromatography mixed-mode C18-DTT stationary phase.

    PubMed

    Wang, Qing; Long, Yao; Yao, Lin; Xu, Li; Shi, Zhi-Guo; Xu, Lanying

    2016-01-01

    A mixed-mode chromatographic stationary phase, C18-DTT (dithiothreitol) silica (SiO2) was prepared through "thiol-ene" click chemistry. The obtained material was characterized by fourier transform infrared spectroscope, nitrogen adsorption analysis and contact angle analysis. Chromatographic performance of the C18-DTT was systemically evaluated by studying the effect of acetonitrile content, pH, buffer concentration of the mobile phase and column temperature. It was demonstrated that the novel stationary phase possessed reversed phase liquid chromatography (RPLC)/hydrophilic interaction liquid chromatography (HILIC) mixed-mode property. The stop-flow test revealed that C18-DTT exhibited excellent compatibility with 100% aqueous mobile phase. Additionally, the stability and column-to-column reproducibility of the C18-DTT material were satisfactory, with relative standard deviations of retention factor of the tested analytes (verapamil, fenbufen, guanine, tetrandrine and nicotinic acid) in the range of 1.82-3.72% and 0.85-1.93%, respectively. Finally, the application of C18-DTT column was demonstrated in the separation of non-steroidal anti-inflammatory drugs, aromatic carboxylic acids, alkaloids, nucleo-analytes and polycyclic aromatic hydrocarbons. It had great resolving power in the analysis of various compounds in HILIC and RPLC chromatographic conditions and was a promising RPLC/HILIC mixed-mode stationary phase.

  14. Deconvoluting the effects of buffer salt concentration in hydrophilic interaction chromatography on a zwitterionic stationary phase.

    PubMed

    West, Caroline; Auroux, Emeline

    2016-08-26

    Quantitative structure-retention relationships (QSRRs) furnish a detailed and reliable description of the role and extent of different molecular interactions that can be established between the analytes and the chromatographic system. Among QSRRs, the solvation parameter model using Abraham descriptors has gained acceptance as a general tool to explore the factors affecting retention in chromatographic systems. We have previously shown how a modified version of the solvation parameter model, with two extra terms to take account of interactions occurring with ionic and ionizable species (with positive and/or negative charges), could be applied to the characterization of hydrophilic interaction chromatographic (HILIC) systems. In the present study, we will show how this methodology can be used to evaluate the effects of increasing buffer salt concentration on retention and separation in a HILIC system. A commercial stationary phase possessing a sulfobetaine zwitterionic bonded ligand (Nucleodur HILIC) was used with a mobile phase composed of 80% acetonitrile and 20% pwwH4 ammonium acetate buffer, with aqueous buffer concentrations varying from 10 to 100mM, resulting in overall concentrations ranging from 2 to 20mM in the mobile phase. Retention factors were measured for a selection of 76 probe analytes. The chosen compounds are small molecules presenting a wide diversity of molecular structures and are relevant to biomedical and pharmaceutical applications. The QSRR models obtained allow for a rationalization of the interactions contributing to retention and separation in the HILIC system considered and shed some light on the effect of varying buffer salt concentration, namely the progressive transition from ion-exchange and electrostatic-repulsion mechanisms to hydrophilic partitioning. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Preparation and characterization of carbon dot-decorated silica stationary phase in deep eutectic solvents for hydrophilic interaction chromatography.

    PubMed

    Zhang, Haijuan; Qiao, Xin; Cai, Tianpei; Chen, Jia; Li, Zhan; Qiu, Hongdeng

    2017-03-01

    In this paper, N-doped carbon dots (NCDs) were successfully decorated on the spherical porous silica surface in deep eutectic solvents (DESs) as a novel class of green solvents. The appropriate density and hydrophility of DESs guaranteed the fine dispersibility of silica particles and NCDs, resulting in a homogeneous and thin layer of NCDs immobilization. As compared with traditional organic solvents (DMF and THF), higher surface coverage was obtained in the medium of DES, proving its feasibility as a new kind of alternative solvent for hydrophilic nanomaterial-based surface modification of silica spheres. The resulting NCDs-decorated silica particles (Sil-NCDs) were characterized in detail and packed into chromatographic columns to study their initial feasibility as adsorbent material for liquid chromatography. The resultant packing materials demonstrate a selective behavior for polar compounds in hydrophilic interaction liquid chromatography (HILIC) mode. This work gives a typical example of using carbon dots as stationary phase component, and such material is hopeful to be used in other research fields such as solid absorbents, recycling catalysts, and solid-state electrochemistry etc. Graphical Abstract N-doped carbon dots (NCDs) were successfully coupled on the surface of porous silica spheres in a green strategy using deep eutectic solvents (DES) as media for HILIC.

  16. Comparison of nonaqueous hydrophilic interaction chromatography with aqueous normal-phase chromatography on hydrosilated silica-based stationary phases.

    PubMed

    Soukup, Jan; Jandera, Pavel

    2013-09-01

    We investigated the retention behavior of phenolic acids in nonaqueous normal-phase (NP) LC with buffered methanol/acetonitrile mobile phases on hydrosilated silica-based stationary phases. The silica hydride, Diamond hydride, Bidentate C18, and Cholesterol columns showed a higher retention of phenolic acids in the nonaqueous mobile phases than in aqueous NP mobile phases. There are some selectivity differences between the aqueous and nonaqueous mobile phases, but generally the resolution and selectivity are better in the aqueous systems. The retention of the phenolic acids tested decreased with increasing concentration of methanol in the mobile phase, up to 20% v/v methanol. At increased temperatures, the retention factors and peak widths decrease in both NP modes, showing linear ln k versus 1/T plots, due to a single retention mechanism over the temperature range from 25°C up to the column stability limit, however, the best separations are achieved at low temperatures. The enthalpic and entropic contributions to the retention were determined, and the differences between the aqueous and nonaqueous modes are possibly due to the adsorbed water layer. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Investigation of the temperature dependence of water adsorption on silica-based stationary phases in hydrophilic interaction liquid chromatography.

    PubMed

    Bartó, Endre; Felinger, Attila; Jandera, Pavel

    2017-03-17

    In the present work, the adsorption of water was investigated in aqueous normal-phase liquid chromatography on Cogent Silica C and Cogent Phenyl hydride stationary phases at different temperatures by frontal analysis - using coulometric Karl Fischer titration - to compare the temperature dependence of adsorption of water from aqueous acetonitrile. The Cogent Silica-C and Cogent Phenyl Hydride columns have a silicon hydride surface (silica hydride) with less than 2% free silanol group; therefore, they do not have a strong association with water. The adsorption behavior of water on the mentioned stationary phases was modeled by Langmuir isotherm. The preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water slightly depends on the temperature. The adsorbed water may fill four to eight percent of the pore volume over the studied temperature range, which approximately corresponds to the equivalent of 0.24-0.68 water layer coverage of the adsorbent surface. The phenyl hydride stationary phase shows decreased water uptake in comparison to the Silica C stationary phase. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Preparation of a novel weak cation exchange/hydrophobic interaction chromatography dual-function polymer-based stationary phase for protein separation using "thiol-ene click chemistry".

    PubMed

    Yang, Fan; Bai, Quan; Zhao, Kailou; Gao, Dong; Tian, Lei

    2015-02-01

    A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) microspheres was synthesized by thiol-ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96% can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2%, 92.4%, and 2.8 × 10(7) IU/mg, respectively, in IEC mode, and 83.4%, 95.2%, and 4.3 × 10(7) IU/mg, respectively, in HIC mode. The results indicate that the

  19. Preparation of a mixed-mode hydrophilic interaction/anion-exchange polymeric monolithic stationary phase for capillary liquid chromatography of polar analytes.

    PubMed

    Lin, Jian; Lin, Jia; Lin, Xucong; Xie, Zenghong

    2009-01-30

    A novel cationic hydrophilic interaction monolithic stationary phase based on the copolymerization of 2-(methacryloyloxy)ethyltrimethylammonium methyl sulfate (META) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was designed for performing capillary liquid chromatography. While META functioned as both the ion-exchange sites and polar ligand provider, the PETA, a trivinyl monomer, was introduced as cross-linker. The monolithic stationary phases with different properties were easily prepared by adjusting the amount of META in the polymerization solution as well as the composition of the porogenic solvent. The hydrophilicity of the monolith increased with increasing content of META in the polymerization mixture. A typical hydrophilic interaction chromatography mechanism was observed when the content of acetonitrile in the mobile phase was higher than 20%. The poly(META-co-PETA) monolith showed very good selectivity for neutral, basic and acidic polar analytes. For polar-charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Peak tailing of basic compounds was avoided and the efficient separation of benzoic acid derivatives was obtained.

  20. The retention behaviour of polar compounds on zirconia based stationary phases under hydrophilic interaction liquid chromatography conditions.

    PubMed

    Kučera, R; Kovaříková, P; Klivický, M; Klimeš, J

    2011-09-28

    The most separations in HILIC mode are performed on silica-based supports. Nevertheless, recently published results have indicated that the metal oxides stationary phases also possess the ability to interact with hydrophilic compounds under HILIC conditions. This paper primarily describes the retention behaviour of model hydrophilic analytes (4-aminobenzene sulfonic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, 3,4-diaminobenzoic acid, 3-aminophenol and 3-nitrophenol) on the polybutadine modified zirconia in HILIC. The results were simultaneously compared with a bare zirconia and a silica-based HILIC phase. The mobile phase strength, pH and the column temperature were systematically modified to assess their impact on the retention of model compounds. It was found that the retention of our model hydrophilic analytes on both zirconia phases was mainly governed by adsorption while on the silica-based HILIC phase partitioning was primarily involved. The ability of ligand-exchange interactions of zirconia surface with a carboxylic moiety influenced substantially the response of carboxylic acids on the elevated temperature as well as to the change of the mobile phase pH in contrast to the silica phase. However, no or negligible ligand-exchange interactions were observed for sulfanilic acid. The results of this study clearly demonstrated the ability of modified zirconia phase to retain polar acidic compounds under HILIC conditions, which might substantially enlarge the application area of the zirconia-based stationary phases. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Stationary phases for packed-column supercritical fluid chromatography.

    PubMed

    Poole, Colin F

    2012-08-10

    The properties of silica-based, chemically bonded, packed column stationary phases used in supercritical fluid chromatography are described with a focus on column design and retention mechanisms. Supercritical fluid chromatography has benefited substantially from innovations in column design for liquid chromatography even if the separation conditions employed are generally quite different. The mobile phase composition and column operating conditions play an interactive role in modifying selectivity in supercritical fluid chromatography by altering analyte solubility in the mobile phase and through selective solvation of the stationary phase resulting in a wider range and intensity of intermolecular interactions with the analyte. The solvation parameter model is used to identify the main parameters that affect retention in supercritical fluid chromatography using carbon dioxide-methanol as a mobile phase and as a basis for column characterization to facilitate the identification of stationary phases with different separation characteristics for method development. As a caution it is pointed out that these column characterization methods are possibly a product of both the stationary phase chemistry and the column operating conditions and are suitable for use only when columns of similar design and with similar operating conditions are used.

  2. Optimization of o-phtaldialdehyde/2-mercaptoethanol postcolumn reaction for the hydrophilic interaction liquid chromatography determination of memantine utilizing a silica hydride stationary phase.

    PubMed

    Douša, Michal; Pivoňková, Veronika; Sýkora, David

    2016-08-01

    A rapid procedure for the determination of memantine based on hydrophilic interaction chromatography with fluorescence detection was developed. Fluorescence detection after postcolumn derivatization with o-phtaldialdehyde/2-mercaptoethanol was performed at excitation and emission wavelengths of 345 and 450 nm, respectively. The postcolumn reaction conditions such as reaction temperature, derivatization reagent flow rate, and reagents concentration were studied due to steric hindrance of amino group of memantine. The derivatization reaction was applied for the hydrophilic interaction liquid chromatography method which was based on Cogent Silica-C stationary phase with a mobile phase consisting of a mixture of 10 mmol/L citric acid and 10 mmol/L o-phosphoric acid (pH 6.0) with acetonitrile using an isocratic composition of 2:8 v/v. The benefit of the reported approach consists in a simple sample pretreatment and a quick and sensitive hydrophilic interaction chromatography method. The developed method was validated in terms of linearity, accuracy, precision, and selectivity according to the International Conference on Harmonisation guidelines. The developed method was successfully applied for the analysis of commercial memantine tablets. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A novel surface-confined glucaminium-based ionic liquid stationary phase for hydrophilic interaction/anion-exchange mixed-mode chromatography.

    PubMed

    Qiao, Lizhen; Wang, Shuangyuan; Li, Hua; Shan, Yuanhong; Dou, Abo; Shi, Xianzhe; Xu, Guowang

    2014-09-19

    Glucaminium-based ionic liquids are a new class of recently developed ionic liquids and prepared by functionalizing the amine group of N-methyl-d-glucamine, which renders them good hydrophilicity due to the presence of the glucose structure and charged quaternary ammonium group. In the present study, a glucaminium-based ionic liquid N,N-diallyl-N-methyl-d-glucaminium bromide was synthesized and subsequently bonded to the surface of 3-mercaptopropyl modified silica materials through "thiol-ene" click chemistry. The obtained stationary phase was characterized by elemental analysis and infrared spectroscopy, and then packed as a HPLC column. A mixture of five nucleosides was used to characterize the separation performance of the obtained column under HILIC mode and the column efficiency was determined with cytidine as the test solute, reaching 80,000plates/m. Then, the retention behavior was evaluated by investigating the effect of various chromatographic factors on retention of different types of solutes, and the results revealed that the developed surface-confined glucaminium-based ionic liquid stationary phase exhibited a hydrophilic interaction/anion-exchange mixed-mode retention mechanism. Finally, two mixtures of nucleotides and flavonoids were separated on the glucaminium-based ionic liquid column, respectively under hydrophilic interaction and hydrophilic interaction/anion-exchange mixed-mode chromatography. In conclusion, the multimodal retention capabilities of the glucaminium-based ionic liquid column could offer a wider range of retention behavior and flexible selectivity toward polar and hydrophilic compounds.

  4. Zirconia--a stationary phase capable of the separation of polar markers of myocardial metabolism in hydrophilic interaction chromatography.

    PubMed

    Kučera, Radim; Kovaříková, Petra; Pasáková-Vrbatová, Ivana; Slaninová, Jitka; Klimeš, Jiří

    2014-05-01

    Creatine, phosphocreatine, and adenine nucleotides are highly polar markers of myocardial metabolism that are poorly retained on RP silica sorbents. Zirconia represents an alternative material to silica with high promise to be used in hydrophilic interaction chromatography (HILIC). This study describes a first systematic investigation of the ability of ZrO2 to separate creatine, phosphocreatine, adenosine 5'-monophosphate, adenosine 5'-diphosphate, and adenosine 5'-triphosphate and compares the results with those obtained on TiO2 . All analytes showed a HILIC-like retention pattern when mobile phases of different strengths were tested. Stronger retention and better column performance were achieved in organic-rich mobile phases as compared to aqueous conditions, where poor retention and insufficient column performance were observed. The effect of mobile phase pH and ionic strength was evaluated as well. The analysis of myocardial tissue demonstrated that all compounds were separated in a relevant biological material and thus proved ZrO2 as a promising phase for HILIC of biological samples that deserves further investigation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Recent development of ionic liquid stationary phases for liquid chromatography.

    PubMed

    Shi, Xianzhe; Qiao, Lizhen; Xu, Guowang

    2015-11-13

    Based on their particular physicochemical characteristics, ionic liquids have been widely applied in many fields of analytical chemistry. Many types of ionic liquids were immobilized on a support like silica or monolith as stationary phases for liquid chromatography. Moreover, different approaches were developed to bond covalently ionic liquids onto the supporting materials. The obtained ionic liquid stationary phases show multi-mode mechanism including hydrophobic, hydrophilic, hydrogen bond, anion exchange, π-π, and dipole-dipole interactions. Therefore, they could be used in different chromatographic modes including ion-exchange, RPLC, NPLC and HILIC to separate various classes of compounds. This review mainly summarizes the immobilized patterns and types of ionic liquid stationary phases, their retention mechanisms and applications in the recent five years.

  6. Synthesis of a reactive polymethacrylate capillary monolith and its use as a starting material for the preparation of a stationary phase for hydrophilic interaction chromatography.

    PubMed

    Kip, Çiğdem; Erkakan, Damla; Gökaltun, Aslıhan; Çelebi, Bekir; Tuncel, Ali

    2015-05-29

    Poly(3-chloro-2-hydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(HPMA-Cl-co-EDMA) capillary monolith was proposed as a reactive starting material with tailoring flexibility for the preparation of monolithic stationary phases. The reactive capillary monolith was synthesized by free radical copolymerization of 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA). The mean pore size, the specific surface area and the permeability of poly(HPMA-Cl-co-EDMA) monoliths were controlled by adjusting porogen/monomer volume ratio, porogen composition and polymerization temperature. The porogen/monomer volume ratio was found as the most effective factor controlling the porous properties of poly(HPMA-Cl-co-EDMA) monolith. Triethanolamine (TEA-OH) functionalized polymethacrylate monoliths were prepared by using the reactive chloropropyl group of poly(HPMA-Cl-co-EDMA) monolith via one-pot and simple post-functionalization process. Poly(HPMA-Cl-co-EDMA) monolith reacted with TEA-OH was evaluated as a stationary phase in nano-hydrophilic interaction chromatography (nano-HILIC). Nucleotides, nucleosides and benzoic acid derivatives were satisfactorily separated with the plate heights up to 20μm. TEA-OH attached-poly(HPMA-Cl-co-EDMA) monolith showed a reproducible and stable retention behaviour in nano-HILIC runs. However, a decrease in the column performance (i.e. an increase in the plate height) was observed with the increasing retention factor. Hence "retention-dependent column efficiency" behaviour was shown for HILIC mode using the chromatographic data collected with the polymer based monolith synthesized.

  7. Monolithic stationary phases with incorporated fumed silica nanoparticles. Part I. Polymethacrylate-based monolithic column with incorporated bare fumed silica nanoparticles for hydrophilic interaction liquid chromatography.

    PubMed

    Aydoğan, Cemil; El Rassi, Ziad

    2016-05-06

    Fumed silica nanoparticles (FSNPs), were incorporated for the first time into a polymethacrylate monolithic column containing glyceryl monomethacrylate (GMM) and ethylene dimethacrylate (EDMA) in order to develop a new monolithic column for hydrophilic interaction high performance liquid chromatography (HILIC). When compared to poly(GMM-EDMA) monolithic column without FSNPs, the same monolithic column with incorporated FSNPs yielded important effects on HILIC separations. The effects of monomers and FSNPs content of the polymerization mixture on the performance of the monolithic column were examined in details, and the optimized stationary phase was investigated over a wide range of mobile phase composition with polar acidic, weakly basic and neutral analytes including hydroxy benzoic acids, nucleotides, nucleosides, dimethylformamide, formamide and thiourea. The retention of these analytes was mainly controlled by hydrophilic interactions with the FSNPs and electrostatic repulsion from the negatively charged silica surface in the case of hydroxy benzoic acids and nucleotides. The electrostatic repulsion was minimized by decreasing the pH of the aqueous component of the mobile phase, which in turn enhanced the retention of acidic solutes. Nucleotides were best separated using step gradient elution at decreasing pH as well as ACN concentration in the mobile phase. Improved peak shape and faster analysis of nucleosides were attained by a fast linear gradient elution with a shallow decrease in the ACN content of the ACN-rich mobile phase. The run-to-run and column-to-column reproducibility were satisfactory. The percent relative standard deviations (%RSDs) for the retention times of tested solutes were lower than 2.5% under isocratic conditions and lower than 3.5 under gradient conditions.

  8. Freeze drying for gas chromatography stationary phase deposition

    DOEpatents

    Sylwester, Alan P.

    2007-01-02

    The present disclosure relates to methods for deposition of gas chromatography (GC) stationary phases into chromatography columns, for example gas chromatography columns. A chromatographic medium is dissolved or suspended in a solvent to form a composition. The composition may be inserted into a chromatographic column. Alternatively, portions of the chromatographic column may be exposed or filled with the composition. The composition is permitted to solidify, and at least a portion of the solvent is removed by vacuum sublimation.

  9. A 2H nuclear magnetic resonance study of the state of water in neat silica and zwitterionic stationary phases and its influence on the chromatographic retention characteristics in hydrophilic interaction high-performance liquid chromatography.

    PubMed

    Wikberg, Erika; Sparrman, Tobias; Viklund, Camilla; Jonsson, Tobias; Irgum, Knut

    2011-09-23

    2H NMR has been used as a tool for probing the state of water in hydrophilic stationary phases for liquid chromatography at temperatures between -80 and +4 °C. The fraction of water that remained unfrozen in four different neat silicas with nominal pore sizes between 60 and 300 Å, and in silicas with polymeric sulfobetaine zwitterionic functionalities prepared in different ways, could be determined by measurements of the line widths and temperature-corrected integrals of the 2H signals. The phase transitions detected during thawing made it possible to estimate the amount of non-freezable water in each phase. A distinct difference was seen between the neat and modified silicas tested. For the neat silicas, the relationship between the freezing point depression and their pore size followed the expected Gibbs-Thomson relationship. The polymeric stationary phases were found to contain considerably higher amounts of non-freezable water compared to the neat silica, which is attributed to the structural effect that the sulfobetaine polymers have on the water layer close to the stationary phase surface. The sulfobetaine stationary phases were used alongside the 100 Å silica to separate a number of polar compounds in hydrophilic interaction (HILIC) mode, and the retention characteristics could be explained in terms of the surface water structure, as well as by the porous properties of the stationary phases. This provides solid evidence supporting a partitioning mechanism, or at least of the existence of an immobilized layer of water into which partitioning could be occurring.

  10. Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability

    PubMed Central

    2016-01-01

    Porous graphitic carbon (PGC) is an important tool in a chromatographer’s armory that retains polar compounds with mass spectrometry (MS)-compatible solvents. However, its applicability is severely limited by an unpredictable loss of retention, which can be attributed to contamination. The solutions offered fail to restore the original retention and our observations of retention time shifts of gemcitabine/metabolites on PGC are not consistent with contamination. The mobile phase affects the ionization state of analytes and the polarizable PGC surface that influences the strength of dispersive forces governing retention on the stationary phase. We hypothesized that failure to maintain the same PGC surface before and after running a gradient is a cause of the observed retention loss/variability on PGC. Herein, we optimize the choice of mobile phase solvent in a gradient program with three parts: a preparatory phase, which allows binding of analytes to column; an elution phase, which gives the required separation/peak shape; and a maintenance phase, to preserve the required retention capacity. Via liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of gemcitabine and its metabolites extracted from tumor tissue, we demonstrate reproducible chromatography on three PGC columns of different ages. This approach simplifies use of the PGC to the same level as that of a C-18 column, removes the need for column regeneration, and minimizes run times, thus allowing PGC columns to be used to their full potential. PMID:27228284

  11. Novel stationary phases based on asphaltenes for gas chromatography.

    PubMed

    Boczkaj, Grzegorz; Momotko, Malwina; Chruszczyk, Dorota; Przyjazny, Andrzej; Kamiński, Marian

    2016-07-01

    We present the results of investigations on the possibility of the application of the asphaltene fraction isolated from the oxidized residue from vacuum distillation of crude oil as a stationary phase for gas chromatography. The results of the investigation revealed that the asphaltene stationary phases can find use for the separation of a wide range of volatile organic compounds. The experimental values of Rohrschneider/McReynolds constants characterize the asphaltenes as stationary phases of medium polarity and selectivity similar to commercially available phases based on alkyl phthalates. Isolation of asphaltenes from the material obtained under controlled process conditions allows the production of a stationary phase having reproducible sorption properties and chromatographic columns having the same selectivity. Unique selectivity and high thermal stability make asphaltenes attractive as a material for stationary phases for gas chromatography. A low production cost from a readily available raw material (oxidized petroleum bitumens) is an important economic factor in case of application of the asphaltene stationary phases for preparative and process separations.

  12. Hydrothermal carbonaceous sphere based stationary phase for anion exchange chromatography.

    PubMed

    Zhao, Qiming; Wu, Shuchao; Zhang, Peimin; Zhu, Yan

    2017-01-15

    Monodisperse carbonaceous spheres produced by the hydrothermal carbonization of sucrose were first applied as green stationary phase for ion chromatography after quaternization. Depending on the polycondensation of methylamine and 1,4-butanediol diglycidyl ether, polymer containing quaternary ammonium groups were facilely grafted onto the surfaces of hydrothermal carbonaceous spheres (HCSs). The quaternized HCSs with different number of polyelectrolyte layers were characterized by scanning electron microscopy, brunauer-emmett-teller, fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis and elemental analysis. The measurements of breakthrough curves demonstrated that more layers of grafted polyelectrolyte resulted in higher anion exchange capacity of stationary phase. With good stability, common inorganic anions, monocarboxylic acids, polarizable anions and carbohydrates were effectively separated on the stationary phases, respectively. The high hydrophilicity of HCS surface afforded excellent peak symmetry for all analytes. Furthermore, high-capacity HCSs stationary phase was successfully applied to detect fluoride in tea samples.

  13. Gold nanoparticle decorated graphene oxide/silica composite stationary phase for high-performance liquid chromatography.

    PubMed

    Liang, Xiaojing; Wang, Xusheng; Ren, Haixia; Jiang, Shengxiang; Wang, Licheng; Liu, Shujuan

    2014-06-01

    In the initial phase of this study, graphene oxide (GO)/silica was fabricated by assembling GO onto the silica particles, and then gold nanoparticles (GNPs) were used to modify the GO/silica to prepare a novel stationary phase for high-performance liquid chromatography. The new stationary phase could be used in both reversed-phase chromatography and hydrophilic interaction liquid chromatography modes. Good separations of alkylbenzenes, isomerides, amino acids, nucleosides, and nucleobases were achieved in both modes. Compared with the GO/silica phase and GNPs/silica phase, it is found that except for hydrophilicity, large π-electron systems, hydrophobicity, and coordination functions, this new stationary phase also exhibited special separation performance due to the combination of 2D GO with zero-dimensional GNPs.

  14. Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model. III. Polar stationary phases.

    PubMed

    West, C; Lesellier, E

    2006-03-31

    In this third paper, varied types of polar stationary phases, namely silica gel (SI), cyano (CN)- and amino-propyl (NH2)-bonded silica, propanediol-bonded silica (DIOL), poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), were investigated in subcritical fluid mobile phase. This study was performed to provide a greater knowledge of the properties of these phases in SFC, and to allow a more rapid and efficient choice of polar stationary phase in regard of the chemical nature of the solutes to be separated. The effect of the nature of the stationary phase on interactions between solute and stationary phases and between solute and carbon dioxide-modifier mobile phases was studied by the use of a linear solvation energy relationship (LSER), the solvation parameter model. The retention behaviour observed with sub/supercritical fluid with carbon dioxide-methanol is close to the one reported in normal-phase liquid chromatography with hexane. The hydrogen bond acidity and basicity, and the polarity/polarizability favour the solute retention when the molar volume of the solute reduces it. As with non-polar phases, the absence of water in the subcritical fluid allows the solute/stationary phase interactions to play a greater part in the retention behaviour. As expected, the DIOL phase and the bare silica display a similar behaviour towards acidic and basic solutes, when interactions with basic compounds are lower with the NH2 phase. On the CN phase, all interactions (hydrogen bonding, dipole-dipole and charge transfer) have a nearly equivalent weight on the retention. The polymeric phases, PEG and PVA, provide the most accurate models, possibly due to their better surface homogeneity.

  15. Silica, hybrid silica, hydride silica and non-silica stationary phases for liquid chromatography.

    PubMed

    Borges, Endler M

    2015-04-01

    Free silanols on the surface of silica are the "villains", which are responsible for detrimental interactions of those compounds and the stationary phase (i.e., bad peak shape, low efficiency) as well as low thermal and chemical stability. For these reasons, we began this review describing new silica and hybrid silica stationary phases, which have reduced and/or shielded silanols. At present, in liquid chromatography for the majority of analyses, reversed-phase liquid chromatography is the separation mode of choice. However, the needs for increased selectivity and increased retention of hydrophilic bases have substantially increased the interest in hydrophilic interaction chromatography (HILIC). Therefore, stationary phases and this mode of separation are discussed. Then, non-silica stationary phases (i.e., zirconium oxide, titanium oxide, alumina and porous graphitized carbon), which afford increased thermal and chemical stability and also selectivity different from those obtained with silica and hybrid silica, are discussed. In addition, the use of these materials in HILIC is also reviewed. © Crown copyright 2014.

  16. A new approach to bioanalysis: aqueous normal-phase chromatography with silica hydride stationary phases.

    PubMed

    Pesek, Joseph J; Matyska, Maria T

    2012-04-01

    Stationary phases based on silica hydride have demonstrated a number of unique properties that are especially advantageous for bioanalyses. They have excellent retention capabilities for hydrophilic compounds, which have been the most difficult to analyze by standard reversed-phase methods and, in many cases, can outperform newer approaches for the analysis of polar molecules, such as hydrophilic liquid interaction chromatography. In addition, all columns utilizing silica-hydride materials can be used in either the normal-phase or reversed-phase modes, sometimes retaining both polar and nonpolar compounds simultaneously. These stationary phases have a high degree of reproducibility and long-term stability.

  17. Silica-based 2-(N,N-dimethylamino)-1,3-propanediol hydrophilic interaction liquid chromatography stationary phase for separating cephalosporins and carbapenems.

    PubMed

    Yin, Wei; Cheng, Lingping; Chai, Huihui; Guo, Ruiqiang; Liu, Renhua; Chu, Changhu; Palasota, John A; Cai, Xiaohui

    2015-08-01

    A silica-based stationary phase bearing both hydrophilic hydroxyl and amino groups was developed by covalently bonding a small molecular N,N-dimethylamino 1,3-propanediol moiety onto silica beads via copper(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition (CuAAC). This new stationary phase showed good HILIC characteristics and high column efficiency (the theoretical plate number is up to 37000 plates m(-1) in the case of inosine) in the separation of polar compounds, such as nucleosides and bases, organic acids, cephalosporins, and carbapenems.

  18. Hierarchical CaCO3 chromatography: a stationary phase based on biominerals.

    PubMed

    Sato, Kosuke; Oaki, Yuya; Takahashi, Daisuke; Toshima, Kazunobu; Imai, Hiroaki

    2015-03-23

    In biomineralization, acidic macromolecules play important roles for the growth control of crystals through a specific interaction. Inspired by this interaction, we report on an application of the hierarchical structures in CaCO3 biominerals to a stationary phase of chromatography. The separation and purification of acidic small organic molecules are achieved by thin-layer chromatography and flash chromatography using the powder of biominerals as the stationary phase. The unit nanocrystals and their oriented assembly, the hierarchical structure, are suitable for the adsorption site of the target organic molecules and the flow path of the elution solvents, respectively. The separation mode is ascribed to the specific adsorption of the acidic molecules on the crystal face and the coordination of the functional groups to the calcium ions. The results imply that a new family of stationary phase of chromatography can be developed by the fine tuning of hierarchical structures in CaCO3 materials. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Surface modification of polytetrafluoroethylene column for two-stationary phase separations by counter-current chromatography.

    PubMed

    Quan, Kai-jun; Huang, Xin-yi; Li, Xiao-ting; Wang, Gao-hong; Liu, Yan-juan; Duan, Wen-da; Di, Duo-long

    2015-11-27

    To improve the separation capability of CCC, a novel solid-liquid two-stationary phases CCC (ASP-CCC) column was prepared employing graphene oxide (GO) conjugated poly-dopamine (PD) coating (GO/PD) as auxiliary stationary phase (ASP). The results of Scanning electron microscopy (SEM), contact angle and X-ray photoelectron spectroscopy (XPS) indicated that nanostructured GO and PD were successfully grafted on the inner wall of the PTFE column. Three alkaloid compounds were selected as the target analytes to evaluate the performance of the novel column. Because of the intermolecular force (hydrogen bond, electrostatic interaction and π-π interaction) between the ASP and model compounds, three analytes were well separated with this novel ASP-CCC column. Additionally, the novel column exhibited higher stationary phase retention ratio, about 8%, than original column without changing the chromatographic condition. Furthermore, the eluotropic sequence of analytes on novel column was in accordance with that in the original column. This suggested that the novel column is a CCC column with auxiliary stationary phase (ASP) in its own right, and the present separation mode is the combination of partition chromatography and adsorption chromatography.

  20. Ionic liquids as novel stationary phases in gas liquid chromatography: inverse or normal isotope effect?

    PubMed

    Schmarr, Hans-Georg; Slabizki, Petra; Müntnich, Sabrina; Metzger, Carmen; Gracia-Moreno, Elisa

    2012-12-28

    The separation of deuterated and non-deuterated compounds in gas liquid partitioning chromatography (GLC) on silicone type stationary phase usually results in the inverse isotope effect. With ionic liquids (ILs) as stationary phase, however, this may show a totally different nature. The inverse isotope effect, in which heavier (deuterated) isotopic compounds (isotopologues) elute earlier, is to be expected when van der Waals (London) dispersion forces play a dominant role in the solute-stationary phase interaction. Such (apolar) interactions seem to play only a minor role when ILs are the stationary phases, leading to only a marginal inverse isotope effect, e.g. for the separation of 2,4,6-trichloroanisole and its [(2)H(5)]-isotopologue on 1,12-di(tripropylphosphonium) dodecane bis(trifluoromethansulfonyl) amide (commercialized as SLB-IL59, Supelco). Indeed, with the most polar stationary phase available (commercialized as SLB-IL111; Supelco), this separation showed a normal isotope effect. Further examples are presented and the nature of the isotope effect observed is discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Enantioselective supercritical fluid chromatography using ristocetin A chiral stationary phases.

    PubMed

    Svensson, L A; Owens, P K

    2000-06-01

    Racemic mixtures of five acidic drugs have been successfully separated by supercritical fluid chromatography (SFC) using macrocyclic antibiotic chiral stationary phases (CSPs). A ristocetin A CSP has been prepared 'in-house' and effectively applied in packed capillary SFC to separate the enantiomers of dichlorprop (R(s) = 1.4), ketoprofen (R(s) = 0.9) and warfarin (R(s) = 0.9). The commercial ristocetin A CSP (Chirobiotic R) was subsequently studied in packed column SFC with similar results where the enantiomers of warfarin (R(s) = 2.2), coumachlor (R(s) = 2.5) and thalidomide (R(s) = 0.6) were separated. Interestingly, differences were observed between the two differently immobilised CSPs where the enantiomers of dichlorprop and ketoprofen, which were separated on the 'in-house' CSP, could not be separated on the commercial phase.

  2. Stationary phase optimized selectivity liquid chromatography: Basic possibilities of serially connected columns using the "PRISMA" principle.

    PubMed

    Nyiredy, Sz; Szucs, Zoltán; Szepesy, L

    2007-07-20

    A new procedure (stationary phase optimized selectivity liquid chromatography: SOS-LC) is described for the optimization of the HPLC stationary phase, using serially connected columns and the principle of the "PRISMA" model. The retention factors (k) of the analytes were determined on three different stationary phases. By use of these data the k values were predicted applying theoretically combined stationary phases. These predictions resulted in numerous intermediate theoretical separations from among which only the optimal one was assembled and tested. The overall selectivity of this separation was better than that of any individual base stationary phase. SOS-LC is independent of the mechanism and the scale of separation.

  3. New oxo-bridged calix[2]arene[2]triazine stationary phase for high performance liquid chromatography.

    PubMed

    Zhao, Wenjie; Hu, Kai; Wang, Caijuan; Liang, Song; Niu, Bailin; He, Lijun; Lu, Kui; Ye, Baoxian; Zhang, Shusheng

    2012-02-03

    A new oxo-bridged calix[2]arene[2]triazine bonded stationary phase (OCATS) for high performance liquid chromatography (HPLC) was prepared using 3-aminopropyltriethoxysilane as coupling reagent. The structure of new material was characterized by infrared spectroscopy, elemental analysis and thermogravimetric analysis. The chromatographic performance and retention mechanism of the new stationary phase were evaluated in reversed-phase mode compared with ODS using different solute probes including polycyclic aromatic hydrocarbons (PAHs), mono-substituted benzenes, disubstituted benzene isomers. The new OCATS stationary phase could provide various interactions for different solutes, such as hydrophobic, hydrogen bonding, ππ and inclusion interactions. The synergistic effects resulting from aromatic rings, bridging oxygen atoms and triazine nitrogen atoms and alkyl linkers in the new material improved the separation selectivity by multiple retention mechanisms. The retention behaviors of the analytes on OCATS column were explained with the assistance of quantum chemistry calculation results using DFT-B3LYP/STO-3G* base group. The OCATS column was successfully employed for the analysis of melamine in infant formula.

  4. Characterization of five chemistries and three particle sizes of stationary phases used in supercritical fluid chromatography.

    PubMed

    Khater, S; West, C; Lesellier, E

    2013-12-06

    Sub-2-microns particles employed as supporting phases are known to favor column efficiency. Recently a set of columns based on sub-2-microns particles for use with supercritical fluid mobile phases have been introduced by Waters. Five different stationary phase chemistries are available: BEH silica, BEHEthyl-pyridine, X Select CSH Fluorophenyl, HSS C18 SB and BEH Shield RP18. This paper describes the characterization of 15 stationary phases, the five different chemistries, and three particle sizes, 1.7 (or 1.8), 3.5 and 5 microns, with the same carbon dioxide–methanol mobile phase and a set of more than a hundred compounds. The interactions established in the 15 different chromatographic systems used in supercritical fluid chromatography (SFC) are assessed with linear solvation energy relationships (LSERs).The results show the good complementarity of the five column chemistries, and their comparative location inside a classification map containing today around 70 different commercial phases. Among the five different chemistries, the HSS C18 SB phase displays a rather unusual behavior in regards of classical C18 phases, as it displays significant hydrogen–bonding interactions. Besides, it appears, as expected, that the BEH Ethyl–pyridine phase has weak interactions with basic compounds. The effect of particle size was studied because smaller particles induce increased inlet and internal pressure. For compressible fluids,this pressure change modifies the fluid density, i.e. the apparent void volume and the eluting strength.These changes could modify the retention and the selectivity of compounds in the case of method trans-fer, by using different particle sizes, from 5 down to 1.7 m. A hierarchical cluster analysis shows that stationary phase clusters were based on the phase chemistry rather than on the particle size, meaning that method transfer from 5 to 1.7 microns can be achieved in the subcritical domain i.e. by using a weakly compressible fluid.

  5. Retention modelling in hydrophilic interaction chromatography.

    PubMed

    Euerby, Melvin R; Hulse, Jennifer; Petersson, Patrik; Vazhentsev, Andrey; Kassam, Karim

    2015-12-01

    The retention behaviour of acidic, basic and quaternary ammonium salts and polar neutral analytes has been evaluated on acidic, basic and neutral hydrophilic interaction chromatography (HILIC) stationary phases as a function of HILIC operating parameters such as MeCN content, buffer concentration, pH and temperature. Numerous empirical HILIC retention models (existing and newly developed ones) have been assessed for their ability to describe retention as a function of the HILIC operating parameters investigated. Retention models have been incorporated into a commercially available retention modelling programme (i.e. ACD/LC simulator) and their accuracy of retention prediction assessed. The applicability of HILIC modelling using these equations has been demonstrated in the two-dimensional isocratic (i.e. buffer concentration versus MeCN content modelling) and one-dimensional gradient separations for a range of analytes of differing physico-chemical properties on the three stationary phases. The accuracy of retention and peak width prediction was observed to be comparable to that reported in reversed-phase chromatography (RPC) retention modelling. Intriguingly, our results have confirmed that the use of gradient modelling to predict HILIC isocratic conditions and vice versa is not reliable. A relative ranking of the importance of the retention and selectivity of HILIC operating parameters has been determined using statistical approaches. For retention, the order of importance was observed to be organic content > stationary phase > temperature ≈ mobile phase pH (i.e. pH 3-6 which mainly effects the ionization of the analyte) ≈ buffer concentration. For selectivity, the nature of the stationary phase > mobile phase pH > buffer concentration > temperature > organic content.

  6. Fluoro-substituted tetraphenyl-phenyl grafted polysiloxanes as highly selective stationary phases for gas chromatography.

    PubMed

    Han, Xue; He, Xinxin; Wang, Huan; Wang, Bing; Wu, Bo

    2016-06-03

    In this work, two new types of polycyclic aromatic grafted polysiloxanes, namely, 3,4-bis(4-fluoro phenyl)-2,5-diphenyl polysiloxane (FPP) and 3,4-bis(3,4,5-trifluoro phenyl)-2,5-diphenyl polysiloxane (TFPP), were synthesized and statically coated onto capillary columns as stationary phases for gas chromatography (GC). Based on their McReynolds constants, both columns exhibited moderate polarity. The efficiencies of the FPP and TFPP columns were 3316 (k=3.96, naphthalene; 0.25mm inner diameter) and 3768 (k=4.14, naphthalene; 0.25mm inner diameter) plates/m, respectively. The thermostability of the polymers was tested by thermogravimetric analysis (TGA), and results revealed that both TFPP and FPP began to decompose slightly at 380°C. Separation of polyethylene pyrolysis products showed that the upper working temperature of the two columns can reach up to 360°C. Relying on their unique polarizable characteristics in combination with other types of interactions, such as H-bond acceptor, dipole-dipole, and dispersive interactions, the newly synthesized polarizable stationary phases offered unique selectivity for aromatic isomers and substituted benzenes. A slight separation difference between TPP and TFPP was observed. TFPP also exerted excellent selectivity for polycyclic aromatic hydrocarbons, fatty acid esters, and fatty alcohols. Overall, FPP and TFPP demonstrated considerable potential for further applications because of their unique structures and outstanding separation performance. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Comparison of liquid and supercritical fluid chromatography mobile phases for enantioselective separations on polysaccharide stationary phases.

    PubMed

    Khater, Syame; Lozac'h, Marie-Anne; Adam, Isabelle; Francotte, Eric; West, Caroline

    2016-10-07

    Analysis and production of enantiomerically pure compounds is a major topic of interest when active pharmaceutical ingredients are concerned. Enantioselective chromatography has become a favourite both at the analytical and preparative scales. High-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) are dominating the scene and are often seen as complementary techniques. Nowadays, for economic and ecologic reasons, SFC may be preferred over normal-phase HPLC (NPLC) as it allows significant reductions in solvent consumption. However, the transfer of NPLC methods to SFC is not always straightforward. In this study, we compare the retention of achiral molecules and separation of enantiomers under supercritical fluid (carbon dioxide with ethanol or isopropanol) and liquid normal-phase (heptane with ethanol or isopropanol) elution modes with polysaccharide stationary phases in order to explore the differences between the retention and enantioseparation properties between the two modes. Chemometric methods (namely quantitative structure-retention relationships and discriminant analysis) are employed to compare the results obtained on a large set of analytes (171 achiral probes and 97 racemates) and gain some understanding on the retention and separation mechanisms. The results indicate that, contrary to popular belief, carbon dioxide - solvent SFC mobile phases are often weaker eluents than liquid mobile phases. It appears that SFC and NPLC elution modes provide different retention mechanisms. While some enantioseparations are unaffected, facilitating the transfer between the two elution modes, other enantioseparations may be drastically different due to different types and strength of interactions contributing to enantioselectivity.

  8. Quinolinium ionic liquid-modified silica as a novel stationary phase for high-performance liquid chromatography.

    PubMed

    Sun, Min; Feng, Juanjuan; Luo, Chuannan; Liu, Xia; Jiang, Shengxiang

    2014-04-01

    A novel stationary phase based on quinolinium ionic liquid-modified silica was prepared and evaluated for high-performance liquid chromatography. The stationary phase was investigated via normal-phase (NP), reversed-phase (RP), and anion-exchange (AE) chromatographic modes, respectively. Polycyclic aromatic hydrocarbons, phthalates, parabens, phenols, anilines, and inorganic anions were used as model analytes in chromatographic separation. Using the newly established column, organic compounds were separated successfully by both NP and RP modes, and inorganic anions were also separated completely by AE mode. The obtained results indicated that the stationary phase could be applied in different chromatographic modes, with multiple-interaction mechanism including van der Waals forces (dipole-dipole, dipole-induced dipole interactions), hydrophobic, π-π stacking, electrostatic forces, hydrogen bonding, anion-exchange interactions, and so on. The column packed with the stationary phase was applied to analyze phthalates and parabens in hexane extracts of plastics. Tap water and bottled water were also analyzed by the column, and nitrate was detected as 20.1 and 13.8 mg L(-1), respectively. The results illustrated that the stationary phase was potential in practical applications.

  9. Mixed Stationary Liquid Phases for Gas-Liquid Chromatography.

    ERIC Educational Resources Information Center

    Koury, Albert M.; Parcher, Jon F.

    1979-01-01

    Describes a laboratory technique for use in an undergraduate instrumental analysis course that, using the interpretation of window diagrams, prepares a mixed liquid phase column for gas-liquid chromatography. A detailed procedure is provided. (BT)

  10. Mixed Stationary Liquid Phases for Gas-Liquid Chromatography.

    ERIC Educational Resources Information Center

    Koury, Albert M.; Parcher, Jon F.

    1979-01-01

    Describes a laboratory technique for use in an undergraduate instrumental analysis course that, using the interpretation of window diagrams, prepares a mixed liquid phase column for gas-liquid chromatography. A detailed procedure is provided. (BT)

  11. Recent progress of chiral stationary phases for separation of enantiomers in gas chromatography.

    PubMed

    Xie, Sheng-Ming; Yuan, Li-Ming

    2017-01-01

    Chromatography techniques based on chiral stationary phases are widely used for the separation of enantiomers. In particular, gas chromatography has developed rapidly in recent years due to its merits such as fast analysis speed, lower consumption of stationary phases and analytes, higher column efficiency, making it a better choice for chiral separation in diverse industries. This article summarizes recent progress of novel chiral stationary phases based on cyclofructan derivatives and chiral porous materials including chiral metal-organic frameworks, chiral porous organic frameworks, chiral inorganic mesoporous materials, and chiral porous organic cages in gas chromatography, covering original research papers published since 2010. The chiral recognition properties and mechanisms of separation toward enantiomers are also introduced.

  12. Development of a decaaza-cyclophane stationary phase for high-performance liquid chromatography.

    PubMed

    Hu, Kai; Deng, Zhifen; Wang, Bei; Cui, Yongxia; Miao, Mingsan; Liu, Wei; Jiang, Qiong; Zhao, Wenjie; Huang, Yanjie; Zhang, Shusheng

    2015-01-01

    A new stationary phase for high-performance liquid chromatography was prepared by covalently bonding a heteroatom-bridged cyclophane onto silica gel using 3-aminopropyltriethoxysilane as the coupling reagent. The structure of the new material was characterized by infrared spectroscopy, elemental analysis, and thermogravimetric analysis. The linear solvation energy relationship method was successfully employed to evaluate the new phase with a set of 25 solutes, and compared with octadecylsilyl and p-tert-butyl-calix[4]arene bonded stationary phases. The retention characteristics of the new phase are similar to the octadecylsilyl and conventional calixarene phases, and it also has distinctive features. In addition, the chromatographic behavior of the phase was illustrated by eluting alkylbenzenes and inorganic anions in the reversed-phase mode and anion-exchange mode, respectively. Thus, multi-interaction mechanisms and mixed-mode separation of the new phase can very likely guarantee its promising application in the analysis of complex samples. The column has been successfully employed for the analysis of triazines in milk, and it is demonstrated to be a competitive alternative analytical method for the determination of triazine herbicide residues.

  13. Review of stationary phases for microelectromechanical systems in gas chromatography: feasibility and separations.

    PubMed

    Azzouz, I; Vial, J; Thiébaut, D; Haudebourg, R; Danaie, K; Sassiat, P; Breviere, J

    2014-02-01

    This review covers the recent development of stationary phases for chip-based gas chromatography (GC). Portable systems for rapid and reliable analysis are urgently needed. One way to achieve this is to miniaturize the entire analysis. Because the column is the central component of the GC system and determines the feasibility and quality of separation, this review focuses on stationary phases reported in the literature and their use in different fields during the last two decades, with emphasis on different methods for introducing the stationary phase into the GC column.

  14. Poly(ethylene oxide)-bonded stationary phase for separation of inorganic anions in capillary ion chromatography.

    PubMed

    Linda, Roza; Lim, Lee Wah; Takeuchi, Toyohide

    2013-06-14

    A tosylated-poly(ethylene oxide) (PEO) reagent was reacted with primary amino groups of an aminopropylsilica packing material (TSKgel NH2-60) in acetonitrile to form PEO-bonded stationary phase. The reaction was a single and simple step reaction. The prepared stationary phase was able to separate inorganic anions. The retention behavior of six common inorganic anions on the prepared stationary phase was examined under various eluent conditions in order to clarify its separation/retention mechanism. The elution order of the tested anions was iodate, bromate, bromide, nitrate, iodide, and thiocyanate, which was similar as observed in common ion chromatography. The retention of inorganic anions could be manipulated by ion exchange interaction which is expected that the eluent cation is coordinated among the PEO chains and it works as the anion-exchange site. Cations and anions of the eluent therefore affected the retention of sample anions. We demonstrated that the retention of the analyte anions decreased with increasing eluent concentration. The repeatability of retention time for the six anions was satisfactory on this column with relative standard deviation values from 1.1 to 4.3% when 10mM sodium chloride was used as the eluent. Compared with the unmodified TSKgel NH2-60, the prepared stationary phase retained inorganic anions more strongly and the selectivity was also improved. The present stationary phase was applied for the determination of inorganic anions contained in various water samples.

  15. Planar gas chromatography column on aluminum plate with multi-walled carbon nanotubes as stationary phase

    NASA Astrophysics Data System (ADS)

    Platonov, I. A.; Platonov, V. I.; Pavelyev, V. S.

    2016-04-01

    The high selectivity of the adsorption layer for low-boiling alkanes is shown, the separation factor (α) couple iso-butane / butane is 1.9 at a column temperature of 50 °C.The paper presents sorption and selective properties of planar gas chromatography column on aluminum plate with multi-walled carbon nanotubes as the stationary phase.

  16. Metal-Organic Framework Thin Films as Stationary Phases in Microfabricated Gas-Chromatography Columns.

    SciTech Connect

    Read, Douglas; Sillerud, Colin Halliday

    2016-01-01

    The overarching goal of this project is to integrate Sandia's microfabricated gas-chromatography ( GC) columns with a stationary phase material that is capable of retaining high-volatility chemicals and permanent gases. The successful integration of such a material with GCs would dramatically expand the repertoire of detectable compounds for Sandia's various microanalysis systems. One such promising class of candidate materials is metal-organic frameworks (MOFs). In this report we detail our methods for controlled deposition of HKUST-1 MOF stationary phases within GC columns. We demonstrate: the chromatographic separation of natural gas; a method for determining MOF film thickness from chromatography alone; and the first-reported GC x GC separation of natural gas -- in general -- let alone for two disparate MOF stationary phases. In addition we determine the fundamental thermodynamic constant for mass sorption, the partition coefficient, for HKUST-1 and several light hydrocarbons and select toxic industrial chemicals.

  17. Sputtered alumina as a novel stationary phase for micro machined gas chromatography columns.

    PubMed

    Haudebourg, R; Matouk, Z; Zoghlami, E; Azzouz, I; Danaie, K; Sassiat, P; Thiebaut, D; Vial, J

    2014-02-01

    Silica and graphite sputtering have previously been reported as novel solid stationary phase deposition techniques for micro gas chromatography columns. As a conventional solid stationary phase in gas chromatography, compatible with sputtering yet so far unreported, alumina was evaluated in this study. Alumina sputtered semi-packed micro columns were fabricated (including an activation step) and proved able to separate a mixture of volatile alkanes (C1-C4 with isomers) in less than 1 min. Kinetic and a thermodynamic evaluation led to calculation of 4,500 theoretical plates for ethane in 1.1 m (HETPmin = 250 μm) and a Gibbs free energy for propane of 30.2 kJ mol(-1), making this stationary phase's properties very close to those observed with silica-sputtered micro columns.

  18. Multi-mode application of graphene quantum dots bonded silica stationary phase for high performance liquid chromatography.

    PubMed

    Wu, Qi; Sun, Yaming; Zhang, Xiaoli; Zhang, Xia; Dong, Shuqing; Qiu, Hongdeng; Wang, Litao; Zhao, Liang

    2017-04-07

    Graphene quantum dots (GQDs), which possess hydrophobic, hydrophilic, π-π stacking and hydrogen bonding properties, have great prospect in HPLC. In this study, a novel GQDs bonded silica stationary phase was prepared and applied in multiple separation modes including normal phase, reversed phase and hydrophilic chromatography mode. Alkaloids, nucleosides and nucleobases were chosen as test compounds to evaluate the separation performance of this column in hydrophilic chromatographic mode. The tested polar compounds achieved baseline separation and the resolutions reached 2.32, 4.62, 7.79, 1.68 for thymidine, uridine, adenosine, cytidine and guanosine. This new column showed satisfactory chromatographic performance for anilines, phenols and polycyclic aromatic hydrocarbons in normal and reversed phase mode. Five anilines were completely separated within 10min under the condition of mobile phase containing only 10% methanol. The effect of water content, buffer concentration and pH on chromatographic separation was further investigated, founding that this new stationary phase showed a complex retention mechanism of partitioning, adsorption and electrostatic interaction in hydrophilic chromatography mode, and the multiple retention interactions such as π-π stacking and π-π electron-donor-acceptor interaction played an important role during the separation process. This GQDs bonded column, which allows us to adjust appropriate chromatography mode according to the properties of analytes, has possibility in actual application after further research.

  19. Preparation and evaluation of surface-bonded tricationic ionic liquid silica as stationary phases for high-performance liquid chromatography.

    PubMed

    Qiao, Lizhen; Shi, Xianzhe; Lu, Xin; Xu, Guowang

    2015-05-29

    Two tricationic ionic liquids were prepared and then bonded onto the surface of supporting silica materials through "thiol-ene" click chemistry as new stationary phases for high-performance liquid chromatography. The obtained columns of tricationic ionic liquids were evaluated respectively in the reversed-phase liquid chromatography (RPLC) mode and hydrophilic interaction liquid chromatography (HILIC) mode, and possess ideal column efficiency of 80,000 plates/m in the RPLC mode with naphthalene as the test solute. The tricationic ionic liquid stationary phases exhibit good hydrophobic and shape selectivity to hydrophobic compounds, and RPLC retention behavior with multiple interactions. In the HILIC mode, the retention and selectivity were evaluated through the efficient separation of nucleosides and bases as well as flavonoids, and the typical HILIC retention behavior was demonstrated by investigating retention changes of hydrophilic solutes with water volume fraction in mobile phase. The results show that the tricationic ionic liquid columns possess great prospect for applications in analysis of hydrophobic and hydrophilic samples.

  20. Synthesis and purification of iodoaziridines involving quantitative selection of the optimal stationary phase for chromatography.

    PubMed

    Boultwood, Tom; Affron, Dominic P; Bull, James A

    2014-05-16

    The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 °C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 °C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control. Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by (1)H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by (1)H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to

  1. [Separation of enantiomers by supercritical fluid chromatography on polysaccharide derivative-based chiral stationary phases].

    PubMed

    Li, Dongyan; Wu, Xi; Hao, Fangli; Yang, Yang; Chen, Xiaoming

    2016-01-01

    Eleven kinds of chiral compounds have been well separated within 10 min on polysaccharide derivative-based chiral stationary phases named Chiralpak IA, IB, IC, ID, IE and IF by supercritical fluid chromatography (SFC). The chiral recognition of these chiral compounds has demonstrated good complementary enantioselectivities of the six chiral columns, which were proved to be useful for chiral SFC. Both the elution time and enantioselectivies could be significantly affected by the modifier types and their concentrations, such as methanol, ethanol and isopropanol, which should be optimized during the experiments. In addition, the solvent versatility of the immobilized chiral stationary phase on the optimization of the chiral separation was helpful.

  2. Synthesis of novel glucose-based polymers and their applications as chiral stationary phases for high performance liquid chromatography.

    PubMed

    Ikai, Tomoyuki; Yamada, Takayuki

    2016-01-01

    Two novel polymers containing glucose units as the main-chain that only differ in terms of their regioregularity were synthesized to evaluate their chiral recognition abilities as chiral stationary phases (CSPs) for high performance liquid chromatography (HPLC). The regioregular polymer (poly-5) shows clear resolution ability for the racemate of cobalt (III) acetylacetonate (Co(acac)3), whereas the corresponding regioirregular polymer (poly-3) does not show any chiral recognition for Co (acac)3. The regioregular polymer main-chain seems to play an important role not only in providing an efficient interaction with the racemate but also in expressing the chiral recognition ability as a CSP for HPLC.

  3. Polar silica-based stationary phases. Part I - Singly and doubly layered sorbents consisting of TRIS-silica and chondroitin sulfate A-TRIS-silica for hydrophilic interaction liquid chromatography.

    PubMed

    Rathnasekara, Renuka; El Rassi, Ziad

    2017-06-01

    Two polar silica bonded stationary phases were prepared by first functionalizing the silica surface with γ-glycidoxypropyltrimethoxysilane, which was then reacted with TRIS to yield a polyhydroxy surface that also has secondary amine functionalities. This step produced the singly layered TRIS-silica column, a cationic hydrophilic column. The TRIS-silica surface was further coated with a layer of chondroitin sulfate A (CSA) yielding the doubly layered hydrophilic CSA-TRIS-silica column. The adsorbed CSA layer provided enhanced hydrophilicity and multi-mode interactions with polar solutes leading to different retention behavior and selectivity compared to the singly layered TRIS-silica column. The anionic sulfate and carboxylate groups in the CSA coating are electrostatically attracted by the cationic TRIS-silica surface yielding a relatively stable physically anchored CSA layer under HILIC elution conditions. The CSA-TRIS-silica column exhibited dual cationic and anionic character with mobile phases at pH ∼3.0 and pH > 4.5, respectively. When comparing solute retention observed on both columns under identical elution conditions, the k values of neutral and cationic solutes were significantly higher on the more hydrophilic doubly layered CSA-TRIS-silica column whereas anionic solutes showed lower k values due to the electrostatic repulsion from the CSA layer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Evaluation of a silicon oxynitride hydrophilic interaction liquid chromatography column in saccharide and glycoside separations.

    PubMed

    Wan, Huihui; Sheng, Qianying; Zhong, Hongmin; Guo, Xiujie; Fu, Qing; Liu, Yanfang; Xue, Xingya; Liang, Xinmiao

    2015-05-01

    The retention characteristics of a silicon oxynitride stationary phase for carbohydrate separation were studied in hydrophilic interaction chromatography mode. Four saccharides including mono-, di-, and trisaccharides were employed to investigate the effects of water content and buffer concentration in the mobile phase on hydrophilic interaction liquid chromatography retention. For the tested saccharides, the silicon oxynitride column demonstrated excellent performance in terms of separation efficiency, hydrophilicity, and interesting separation selectivity for carbohydrates compared to the bare silica stationary phase. Finally, the silicon oxynitride hydrophilic interaction liquid chromatography column was employed in the separation of complex samples of fructooligosaccharides, saponins, and steviol glycoside from natural products. The resulting chromatograms demonstrated good separation efficiency and longer retention compared with silica, which further confirmed the advantages and potential application of silicon oxynitride stationary phase for hydrophilic interaction liquid chromatography separation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Performance and selectivity of cationic nanoparticle pseudo-stationary phases in electrokinetic chromatography.

    PubMed

    McGettrick, Julie R; Williamson, Nathan H; Sutton, Adam T; Palmer, Christopher P

    2017-03-01

    Electrokinetic chromatography (EKC) is a powerful analytical technique that uses an ionic pseudo-stationary phase (PSP) to separate neutral compounds. Although anionic surfactants are the most common choice for PSP, cationic latex nanoparticles are an attractive alternative. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize several types of diblock copolymers that self-assemble into latex nanoparticles, which were characterized by a variety of techniques including diffusion NMR. The performance of each nanoparticle as a PSP was studied by using a homologous series of ketones and linear solvation energy relationships (LSER) analysis. A cationic homopolymer coating was found to be necessary to prevent band broadening caused by analyte interactions with nanoparticles adsorbed to the capillary surface. No significant difference in methylene selectivity or LSER parameters was observed between nanoparticles with different cationic shells, but differences were observed between nanoparticles with different hydrophobic cores. Cationic latex nanoparticles behaved more like anionic latex nanoparticles than like cationic surfactants, suggesting that selectivity is primarily driven by the hydrophobic portion of a PSP. Cationic latex nanoparticles in combination with a homopolymer cationic capillary coating are an excellent choice for EKC analyses where an anodic electroosmotic flow is required. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Experimental comparison of chiral metal-organic framework used as stationary phase in chromatography.

    PubMed

    Xie, Sheng-Ming; Zhang, Mei; Fei, Zhi-Xin; Yuan, Li-Ming

    2014-10-10

    Chiral metal-organic frameworks (MOFs) are a new class of multifunctional material, which possess diverse structures and unusual properties such as high surface area, uniform and permanent cavities, as well as good chemical and thermal stability. Their chiral functionality makes them attractive as novel enantioselective adsorbents and stationary phases in separation science. In this paper, the experimental comparison of a chiral MOF [In₃O(obb)₃(HCO₂)(H₂O)] solvent used as a stationary phase was investigated in gas chromatography (GC), high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The potential relationship between the structure and components of chiral MOFs with their chiral recognition ability and selectivity are presented. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Stabilized phospholipid membranes in chromatography: toward membrane protein-functionalized stationary phases.

    PubMed

    Gallagher, Elyssia S; Mansfield, Elisabeth; Aspinwall, Craig A

    2014-04-01

    Transmembrane protein (TMP)-functionalized materials have resulted in powerful new methods in chemical analysis. Of particular interest is the development of high-throughput, TMP-functionalized stationary phases for affinity chromatography of complex mixtures of analytes. Several natural and synthetic phospholipids and lipid mimics have been used for TMP reconstitution, although the resulting membranes often lack the requisite chemical and temporal stability for long-term use, a problem that is exacerbated in flowing separation systems. Polymerizable lipids with markedly increased membrane stability and TMP functionality have been developed over the past two decades. More recently, these lipids have been incorporated into a range of analytical methods, including separation techniques, and are now poised to have a significant impact on TMP-based separations. Here, we describe current methods for preparing TMP-containing stationary phases and examine the potential utility of polymerizable lipids in TMP affinity chromatography.

  8. Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC).

    PubMed

    Huang, Yining; Nie, Yongxin; Boyes, Barry; Orlando, Ron

    2016-09-01

    The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in α2-3 and α2-6 linkages, while these glycans remain attached to peptides.

  9. Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC)

    PubMed Central

    Huang, Yining; Nie, Yongxin; Boyes, Barry

    2016-01-01

    The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in α2-3 and α2-6 linkages, while these glycans remain attached to peptides. PMID:27582638

  10. Coating properties of a novel water stationary phase in capillary supercritical fluid chromatography.

    PubMed

    Murakami, Jillian N; Thurbide, Kevin B

    2015-05-01

    The coating properties of a novel water stationary phase used in capillary supercritical fluid chromatography were investigated. The findings confirm that increasing the length or internal diameter of the type 316 stainless-steel column used provides a linear increase in the volume of stationary phase present. Under normal operating conditions, results indicate that about 4.9 ± 0.5 μL/m of water phase is deposited uniformly inside of a typical 250 μm internal diameter 316 stainless-steel column, which translates to an area coverage of about 6.3 ± 0.5 nL/mm(2) regardless of dimension. Efforts to increase the stationary phase volume present showed that etching the stainless-steel capillary wall using hydrofluoric acid was very effective for this. For instance, after five etching cycles, this volume doubled inside of both the type 304 and the type 316 stainless-steel columns examined. This in turn doubled analyte retention, while maintaining good peak shape and column efficiency. Overall, 316 stainless-steel columns were more resistant to etching than 304 stainless-steel columns. Results indicate that this approach could be useful to employ as a means of controlling the volume of water stationary phase that can be established inside of the stainless-steel columns used with this supercritical fluid chromatography technique.

  11. Recent development in liquid chromatography stationary phases for separation of Traditional Chinese Medicine components.

    PubMed

    Jin, Hongli; Liu, Yanfang; Guo, Zhimou; Wang, Jixia; Zhang, Xiuli; Wang, Chaoran; Liang, Xinmiao

    2016-10-25

    Traditional Chinese Medicine (TCM) is an ancient medical practice which has been used to prevent and cure diseases for thousands of years. TCMs are frequently multi-component systems with mainly unidentified constituents. The study of the chemical compositions of TCMs remains a hotspot of research. Different strategies have been developed to manage the significant complexity of TCMs, in an attempt to determine their constituents. Reversed-phase liquid chromatography (RPLC) is still the method of choice for the separation of TCMs, but has many problems related to limited selectivity. Recently, enormous efforts have been concentrated on the development of efficient liquid chromatography (LC) methods for TCMs, based on selective stationary phases. This can improve the resolution and peak capacity considerably. In addition, high-efficiency stationary phases have been applied in the analysis of TCMs since the invention of ultra high-performance liquid chromatography (UHPLC). This review describes the advances in LC methods in TCM research from 2010 to date, and focuses on novel stationary phases. Their potential in the separation of TCMs using relevant applications is also demonstrated.

  12. Monolayer-Protected Gold Nanoparticles as a Stationary Phase for Open Tubular Gas Chromatography

    SciTech Connect

    Gross, Gwen M.; Nelson, David A.; Grate, Jay W.; Synovec, Robert E.

    2003-09-01

    The use of a thin film of monolayer protected gold nanoparticles (MPNs) as a stationary phase for gas chromatography (GC) is reported. Dodecanethiol-protected gold nanoparticles were prepared and characterized. Deposition of a MPN film was successfully completed within a 2 m, 530 {micro}m (i.d.) deactivated silica capillary using gravity to force a plug of solution containing the MPN material through the capillary for deposition. The presence of a thin MPN film on the GC capillary inside wall was confirmed with SEM analysis with an average film thickness of 60.7 nm measured. The retention behavior of the dodecanethiol MPN stationary phase was studied using four different classes of compounds (alkanes, alcohols, aromatics and ketones) and their retention orders were compared to a commercially available column (AT-1, 100 nm phase thickness). The separation of an eight-component mixture was performed using both isothermal and temperature programming separation methods with the novel dodecanethiol MPN phase. The isothermal separation was then objectively compared to the commercial AT-1 stationary phase column using the same experimental parameters. The commercial column had an efficiency, N, of 6200 (k{prime} = 0.33) while the dodecanethiol MPN stationary phase had an efficiency, N, of 5700 (k{prime} = 0.21) for the same analyte, octane. The reduced plate height, h, for this same analyte was found to be less than 1 at the optimum linear flow velocity. Based upon the efficiencies and reduced plate height studies as a function of linear flow velocity, we conclude that the MPN stationary phase operated at nearly the optimum possible performance level. The robustness of the MPN phase is also discussed with consistent performance observed over several months. Overall, the use of monolayer protected gold nanoparticles as gas chromatographic stationary phase materials appears promising.

  13. High-temperature separations on a polymer-coated fibrous stationary phase in microcolumn liquid chromatography.

    PubMed

    Nakane, Kenichi; Shirai, Shingoro; Saito, Yoshihiro; Moriwake, Yusuke; Ueta, Ikuo; Inoue, Mitsuru; Jinno, Kiyokatsu

    2011-01-01

    Novel polymer-coated fiber-packed microcolumns in liquid chromatography (LC) have been developed. Typical polymeric materials, such as polydimethylsiloxane and polyethyleneglycol, which are conventional stationary phases of capillary columns in gas chromatography (GC), have been employed as coating materials onto the surface of fine filaments. Packed longitudinally with a bundle of polymer-coated filaments into a stainless-steel capillary of 0.8 mm i.d., 150 mm length, several types of polymer-coated fiber-packed columns were prepared, and the retention behavior of aromatic compounds on these columns has been studied. A good linear relationship was obtained for van't Hoff plots over the temperature range between 0 and 200 °C, clearly indicating an excellent heat-resistant property of these polymer-coated fibrous stationary phases. Taking advantage of the heat-resistant feature of the fibrous stationary phases, the separation of several test mixtures with temperature-programmed elution was studied, where a solvent gradient program was additionally introduced if needed. Separation was also carried out with pure water as the mobile phase using an appropriate temperature program. 2011 © The Japan Society for Analytical Chemistry

  14. Determination of solute partition behavior with room-temperature ionic liquid based micellar gas-liquid chromatography stationary phases using the pseudophase model.

    PubMed

    Lantz, Andrew W; Pino, Verónica; Anderson, Jared L; Armstrong, Daniel W

    2006-05-19

    The use of micelles in ionic liquid based gas-chromatography stationary phases was evaluated using equations derived for a "three-phase" model. This model allows the determination of all three partition coefficients involved in the system, and elucidates the micellar contribution to retention and selectivity. Four types of micellar-ionic liquid columns were examined in this study: 1-butyl-3-methylimidazolium chloride with sodium dodecylsulfate or dioctyl sulfosuccinate, and 1-butyl-3-methylimidazolium hexafluorophosphate with polyoxyethylene-100-stearyl ether or polyoxyethylene-23-lauryl ether. The partition coefficients were measured for a wide range of probe molecules capable of a variety of types and magnitudes of interactions. In general, most probe molecules preferentially partitioned to the micellar pseudophase over the bulk ionic liquid component of the stationary phase. Therefore, addition of surfactant to the stationary phase usually resulted in greater solute retention. It is also shown that the selectivity of the stationary phase is significantly altered by the presence of micelles, either by enhancing or lessening the separation. The effects of surfactant on the interaction parameters of the stationary phase are determined using the Abraham solvation parameter model. The addition of sodium dodecylsulfate and dioctyl sulfosuccinate to 1-butyl-3-methylimidazolium chloride stationary phases generally increased the phase's hydrogen bond basicity and increased the level of dispersion interaction. Polyoxyethylene-100-stearyl ether and polyoxyethylene-23-lauryl ether surfactants, however, enhanced the pi-pi/n-pi, polarizability/dipolarity, and hydrogen bond basicity interactions of 1-butyl-3-methylimidazolium hexafluorophosphate to a greater degree than the ionic surfactants with 1-butyl-3-methylimidazolium chloride. However, these nonionic surfactants appeared to hinder the ability of the stationary phase to interact with solutes via dispersion forces

  15. Preparation of a silica stationary phase co-functionalized with Wulff-type phenylboronate and C12 for mixed-mode liquid chromatography.

    PubMed

    Li, Hengye; Zhang, Xuemeng; Zhang, Lin; Wang, Xiaojin; Kong, Fenying; Fan, Dahe; Li, Lei; Wang, Wei

    2017-04-15

    A silica stationary phase was designed and synthesized through the co-functionalization of silica with Wulff-type phenylboronate and C12 for mixed-mode liquid chromatography applications. The as-synthesized stationary phase was characterized by elemental analysis and Fourier Transform-InfraRed Spectroscopy (FT-IR). Retention mechanisms, including boronate affinity (BA), reversed-phase (RP) and anion-exchange (AE), were involved. Retention mechanism switching was easily realized by adjustment of the mobile phase constitution. Cis-diol compounds could be selectively captured under neutral conditions in BA mode and off-line separated in RP mode. Neutral, basic, acidic and amphiprotic compounds were chromatographed on the column in RP chromatography, while inorganic anions were chromatographed in AE chromatography to characterize the mixed-mode nature of the prepared stationary phase. In addition, the RP performance was compared with an octadecyl silica column in terms of column efficiency (N/m), asymmetry factor (Af), retention factor (k) and resolution (Rs). The prepared stationary phase offered multiple interactions with analytes in addition to hydrophobic interactions under RP elution conditions. Based on the mixed-mode properties, off-line 2D-LC, for selective capture and separation of urinary nucleosides, was successfully realized on a single column, demonstrating its powerful application potential for complex samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Sum of ranking differences to rank stationary phases used in packed column supercritical fluid chromatography.

    PubMed

    West, Caroline; Khalikova, Maria A; Lesellier, Eric; Héberger, Károly

    2015-08-28

    The identification of a suitable stationary phase in supercritical fluid chromatography (SFC) is a major source of difficulty for those with little experience in this technique. Several protocols have been suggested for column classification in high-performance liquid chromatography (HPLC), gas chromatography (GC), and SFC. However, none of the proposed classification schemes received general acceptance. A fair way to compare columns was proposed with the sum of ranking differences (SRD). In this project, we used the retention data obtained for 86 test compounds with varied polarity and structure, analyzed on 71 different stationary phases encompassing the full range in polarity of commercial packed columns currently available to the SFC chromatographer, with a single set of mobile phase and operating conditions (carbon dioxide-methanol mobile phase, 25°C, 150bar outlet pressure, 3ml/min). First, a reference column was selected and the 70 remaining columns were ranked based on this reference column and the retention data obtained on the 86 analytes. As these analytes previously served for the calculation of linear solvation energy relationships (LSER) on the 71 columns, SRD ranks were compared to LSER methodology. Finally, an external comparison based on the analysis of 10 other analytes (UV filters) related the observed selectivity to SRD ranking. Comparison of elution orders of the UV filters to the SRD rankings is highly supportive of the adequacy of SRD methodology to select similar and dissimilar columns.

  17. Photochemically Immobilized 4-Methylbenzoyl Cellulose as a Powerful Chiral Stationary Phase for Enantioselective Chromatography.

    PubMed

    Francotte, Eric; Huynh, Dan; Zhang, Tong

    2016-12-17

    A process to immobilize para-methylbenzoyl cellulose (PMBC) on silica gel has been developed and applied to prepare chiral stationary phases (CSPs) for enantioselective chromatography. The immobilization was achieved by simple irradiation of the polysaccharide derivative with ultraviolet light after coating on a silica gel support. The influence of parameters such as irradiation time and solvent on immobilization effectiveness were investigated. The performance of the prepared immobilized phases were evaluated by injection of a series of racemic compounds onto the packed columns and determination of their chiral recognition ability. By contrast to the classical coated phase, the immobilized CSP can be used under various chromatographic conditions without limitation of organic solvent types as the mobile phase. This extended applicability permits to improve selectivity and to resolve chiral compounds which are not or only poorly soluble in the mobile phases which are compatible with the non-immobilized PMBC stationary phase.

  18. Liquid Chromatography with mass spectrometry analysis of mycotoxins in food samples using silica hydride based stationary phases.

    PubMed

    Pesek, Joseph J; Matyska, Maria T; Hoffmann, Jessica F; Madruga, Nathalia A; Crizel, Rosane L; Elias, Moacir C; Vanier, Nathan L; Chaves, Fabio C

    2017-05-01

    Liquid chromatography with mass spectrometry analysis of selected food samples using silica hydride stationary phases allowed for the identification and quantification of common mycotoxins including aflatoxin B1, B2, G1, G2, ochratoxin A, and fumosinin B1. Phenyl and C18 columns showed relatively similar selectivity based on hydrophobicity but the phenyl phase provides an additional mechanism, π-π interaction. The most hydrophobic of the analyzed compounds was more strongly retained on the C18 column and also has fewer unsaturated sites, which limited the interaction with the phenyl phase. Bean, maize, rice, and wheat samples were harvested and stored under conditions conducive to fungal development, and all samples presented toxin contamination exceeding the maximum tolerable limits. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Electrochemically modulated liquid chromatography using a boron-doped diamond particle stationary phase

    PubMed Central

    Muna, Grace W.; Swope, Vernon M.; Swain, Greg M.; Porter, Marc D.

    2011-01-01

    This paper reports on preliminary tests of the performance of boron-doped diamond powder (BDDP) as a stationary phase in electrochemically modulated liquid chromatography (EMLC). EMLC manipulates retention through changes in the potential applied (Eappl) to a conductive packing. Porous graphitic carbon (PGC) has routinely been utilized as a material in EMLC separations. Herein the utility of BDDP as a stationary phase in EMLC was investigated and its stability, both compositionally and microstructurally, relative to PGC was compared. The results show that BDDP is stable over a wide range of Eappl values (i.e., −1.2 to +1.2 V vs. Ag/AgCl, sat’d NaCl). The data also reveal that electrostatics play a key role in the adsorption of the aromatic sulfonates on the BDDP stationary phase, and that these analytes are more weakly retained in comparison to the PGC support. The potential for this methodology to provide a means to advance the understanding of molecular adsorption and retention mechanisms on carbonaceous materials is briefly discussed. PMID:18922535

  20. Homochiral metal-organic framework used as a stationary phase for high-performance liquid chromatography.

    PubMed

    Kong, Jiao; Zhang, Mei; Duan, Ai-Hong; Zhang, Jun-Hui; Yang, Rui; Yuan, Li-Ming

    2015-02-01

    Metal-organic frameworks are promising porous materials. Chiral metal-organic frameworks have attracted considerable attention in controlling enantioselectivity. In this study, a homochiral metal-organic framework [Co(2) (D-cam)(2) (TMDPy)] (D-cam = D-camphorates, TMDPy = 4,4'-trimethylenedipyridine) with a non-interpenetrating primitive cubic net has been used as a chiral stationary phase in high-performance liquid chromatography. It has allowed the successful separation of six positional isomers and six chiral compounds. The good selectivity and baseline separation, or at least 60% valley separation, confirmed its excellent molecular recognition characteristics. The relative standard deviations for the retention time of run-to-run and column-to-column were less than 1.8 and 3.1%, respectively. These results demonstrate that [Co(2) (D-cam)(2) (TMDPy)] may represent a promising chiral stationary phase for use in high-performance liquid chromatography. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Validation of stationary phases in (111)In-pentetreotide planar chromatography.

    PubMed

    Moreno-Ortega, E; Mena-Bares, L M; Maza-Muret, F R; Hidalgo-Ramos, F J; Vallejo-Casas, J A

    2013-01-01

    Since Pall-German stopped manufacturing ITLC-SG, it has become necessary to validate alternative stationary phases. To validate different stationary phases versus ITLC-SG Pall-Gelman in the determination of the radiochemical purity (RCP) of (111)In-pentetreotide ((111)In-Octreoscan) by planar chromatography. We conducted a case-control study, which included 66 (111)In-pentetreotide preparations. We determined the RCP by planar chromatography, using a freshly prepared solution of 0,1M sodium citrate (pH 5) and the following stationary phases: ITLC-SG (Pall-Gelman) (reference method), iTLC-SG (Varian), HPTLC silica gel 60 (Merck), Whatman 1, Whatman 3MM and Whatman 17. For each of the methods, we calculated: PRQ, relative front values (RF) of the radiopharmaceutical and free (111)In, chromatographic development time, resolution between peaks. We compared the results obtained with the reference method. The statistical analysis was performed using the SPSS program. The p value was calculated for the study of statistical significance. The highest resolution is obtained with HPTLC silica gel 60 (Merck). However, the chromatographic development time is too long (mean=33.62minutes). Greater resolution is obtained with iTLC-SG (Varian) than with the reference method, with lower chromatographic development time (mean=3.61minutes). Very low resolutions are obtained with Whatman paper, essentially with Whatman 1 and 3MM. Therefore, we do not recommend their use. Although iTLC-SG (Varian) and HPTLC silica gel 60 (Merck) are suitable alternatives to ITLC-SG (Pall-Gelman) in determining the RCP of (111)In-pentetreotide, iTLC-SG (Varian) is the method of choice due to its lower chromatographic development time. Copyright © 2012 Elsevier España, S.L. and SEMNIM. All rights reserved.

  2. Separation performance of cucurbit[7]uril in ionic liquid-based sol-gel coating as stationary phase for capillary gas chromatography.

    PubMed

    Wang, Xiaogang; Qi, Meiling; Fu, Ruonong

    2014-12-05

    Here we report the separation performance of a new stationary phase of cucurbit[7]uril (CB7) incorporated into an ionic liquid-based sol-gel coating (CB7-SG) for capillary gas chromatography (GC). The CB7-SG stationary phase showed an average polarity of 455, suggesting its polar nature. Abraham system constants revealed that its major interactions with analytes include H-bond basicity (a), dipole-dipole (s) and dispersive (l) interactions. The CB7-SG stationary phase achieved baseline separation for a wide range of analytes with symmetrical peak shapes and showed advantages over the conventional polar stationary phase that failed to resolve some critical analytes. Also, it exhibited different retention behaviors from the conventional stationary phase in terms of retention times and elution order. Most interestingly, in contrast to the conventional polar phase, the CB7-SG stationary phase exhibited longer retentions for analytes of lower polarity but relatively comparable retentions for polar analytes such as alcohols and phenols. The high resolving ability and unique retention behaviors of the CB7-SG stationary phase may stem from the comprehensive interactions of the aforementioned interactions and shape selectivity. Moreover, the CB7-SG column showed good peak shapes for analytes prone to peak tailing, good thermal stability up to 280°C and separation repeatability with RSD values in the range of 0.01-0.11% for intra-day, 0.04-0.41% for inter-day and 2.5-6.0% for column-to-column, respectively. As demonstrated, the proposed coating method can simultaneously address the solubility problem with CBs for the intended purpose and achieve outstanding GC separation performance.

  3. Tubing modifications for countercurrent chromatography (CCC): Stationary phase retention and separation efficiency.

    PubMed

    Englert, Michael; Vetter, Walter

    2015-07-16

    Countercurrent chromatography (CCC) is a separation technique in which two immiscible liquid phases are used for the preparative purification of synthetic and natural products. In CCC the number of repetitive mixing and de-mixing processes, the retention of the stationary phase and the mass transfer between the liquid phases are significant parameters that influence the resolution and separation efficiency. Limited mass transfer is the main reason for peak broadening and a low number of theoretical plates along with impaired peak resolution in CCC. Hence, technical improvements with regard to column design and tubing modifications is an important aspect to enhance mixing and mass transfer. In this study we constructed a crimping tool which allowed us to make reproducible, semi-automated modifications of conventional round-shaped tubing. Six crimped tubing modifications were prepared, mounted onto multilayer coils which were subsequently installed in the CCC system. The stationary phase retention of the tubing modifications were compared to the conventional system with unmodified tubing in a hydrophobic, an intermediate and a hydrophilic two-phase solvent system. Generally, the tubing modifications provided higher capabilities to retain the stationary phase depending on the solvent system and flow rates. In the intermediate solvent system the separation efficiency was evaluated with a mixture of six alkyl p-hydroxybenzoates. The peak resolution could be increased up to 50% with one of the tubing modifications compared to the unmodified tubing. Using the most convincing tubing modification at fixed values for the stationary phase retention, a reasonable comparison to the unmodified tubing was achieved. The peak width could be reduced up to 49% and a strong positive impact at increased flow rates regarding peak resolution and theoretical plate number was observed compared to unmodified tubing. It could be concluded that the tubing modification enhanced the interphase

  4. Selectivity of stationary phases based on pyridinium ionic liquids for capillary gas chromatography

    NASA Astrophysics Data System (ADS)

    Shashkov, M. V.; Sidelnikov, V. N.; Zaikin, P. A.

    2014-04-01

    A number of capillary columns with stationary liquid phases based on mono- and dication pyridinium ionic liquids (ILs) were prepared. Their polarity was evaluated using McReynolds system and the selectivity was estimated from intermolecular interactions. The parameters of intermolecular interactions were obtained from retention data using the (Abraham) model of the linear free energy relationship. The dependences of intermolecular interactions on the structure of the cation in the ILs under study were revealed. The results were compared with the data for the traditional phases (HP-5, ZB-WAX). Examples of separation of mixtures of oxygen-containing compounds on the phases under study are given.

  5. Grain-grain interaction in stationary dusty plasma

    SciTech Connect

    Lampe, Martin; Joyce, Glenn

    2015-02-15

    We present a particle-in-cell simulation study of the steady-state interaction between two stationary dust grains in uniform stationary plasma. Both the electrostatic force and the shadowing force on the grains are calculated explicitly. The electrostatic force is always repulsive. For two grains of the same size, the electrostatic force is very nearly equal to the shielded electric field due to a single isolated grain, acting on the charge of the other grain. For two grains of unequal size, the electrostatic force on the smaller grain is smaller than the isolated-grain field, and the force on the larger grain is larger than the isolated-grain field. In all cases, the attractive shadowing force exceeds the repulsive electrostatic force when the grain separation d is greater than an equilibrium separation d{sub 0}. d{sub 0} is found to be between 6λ{sub D} and 9λ{sub D} in all cases. The binding energy is estimated to be between 19 eV and 900 eV for various cases.

  6. Preparation and characterization of mesoporous silicas modified with chiral selectors as stationary phase for high-performance liquid chromatography.

    PubMed

    Pérez-Quintanilla, Damián; Morante-Zarcero, Sonia; Sierra, Isabel

    2014-01-15

    New hybrid materials were prepared as novel chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). Pure mesoporous silica (SM) and ethylene-bridged periodic mesostructured organosilica (PMO) were functionalized, by a post-synthesis method, with derivates of erythromycin and vancomycin. N2 adsorption-desorption measurements, XRD, FT-IR, MAS NMR, SEM, TEM and elemental analysis were used to characterize the physico-chemical properties of these mesostructured materials, before and after the modification process. The synthesized particles had non-symmetrical 3-D wormhole-like mesostructure, spherical morphology, and a mean pore diameter between 53 and 59 Å. CSPs prepared were tested for the separation of four chiral β-blockers (atenolol, metoprolol, pindolol and propranolol) in normal phase (NP) and polar organic phase (PO) elution modes. Much stronger chiral interaction was observed in vancomycin-modified silicas. Results obtained in these preliminary studies will permit in future works to improve the synthesis route in order to design mesoporous materials with better performance as a chiral stationary phase for HPLC. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. A new methodology to determine the isoeluotropic conditions on ultra-performance flash purification stationary phases from analytical reversed liquid chromatography stationary phase.

    PubMed

    Héron, Sylvie; Charbonneau, Didier; Albisson, Pauline; Estievenart, Guillaume; Groni, Sihem; Tchapla, Alain

    2015-06-05

    Nowadays, the determination of the experimental chromatographic conditions to be used in Reversed Phase Liquid Ultra-Performance Flash Purification is still challenging. This is due to four different items. In most cases, flash purification stationary phases are not available with geometry of column used in analytical chromatography. The flash purification columns are single-use only. From the point of view of selectivity and retention, few RPLC phases exist with properties of separation identical for analytical and flash purification supports. Characterization methods and databases used for comparing analytical RPLC columns do not include stationary phases for RP flash purification columns. The goal of this work is to develop a new method development strategy which permits the determination of the experimental chromatographic conditions on RP ultra-performance flash purification columns. It relies on the knowledge of any isocratic conditions obtained on any given initial reversed stationary phase. The final conditions to implement on the RP ultra-performance flash purification phase enable either to keep the retention range of a selected solute constant, or to set it around a previously chosen value. The rules of transfer in linear gradient mode are also described. The methodology was valid, whatever the initial RP stationary and mobile phases, for different chemical classes, whatever the bonding, particle diameter, porous or core shell particle, towards different RP alkyl and analogues stationary and mobile phases. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Bifurcation analysis of interacting stationary modes in thermohaline convection

    SciTech Connect

    Neveling, M.; Dangelmayr, G.

    1988-09-01

    The Boussinesq equations for thermohaline convection in a finite two-dimensional box and with stress-free boundaries are considered. There are critical values of the aspect ratio at which the conduction state becomes unstable to two different roll patterns simultaneously. Near such a critical value a center manifold reduction allows us to reduce the dynamical behavior of the Boussinesq equations to a standard normal form equation that describes the interaction of two stationary modes. We present explicit analytical expressions for the linear and nonlinear coefficients on which the normal form depends. A numerical investigation of these coefficients leads to a division of the space of parameters (Prandtl number, solute Rayleigh number, Lewis number) into various regions that give rise to qualitatively different bifurcation behavior. Besides those encountered in ordinary convection, a variety of further phenomena is found, in particular in a vicinity of double tricritical points.

  9. Nano-particle modified stationary phases for high-performance liquid chromatography.

    PubMed

    Nesterenko, Ekaterina P; Nesterenko, Pavel N; Connolly, Damian; He, Xiaoyun; Floris, Patrick; Duffy, Emer; Paull, Brett

    2013-08-07

    This review covers the latest developments and applications of nano-materials in stationary phase development for various modes of high-performance liquid chromatography. Specific attention is placed upon the development of new composite phases, including the synthetic and immobilisation strategies used, to produce either encapsulated nano-particles, or surface attached nano-particles, layers, coatings and other structures. The resultant chromatographic applications, where applicable, are discussed with comment upon enhanced selectivity and/or efficiency of the nano-particle modified phases, where such effects have been identified. In the main this review covers developments over the past five years and is structured according to the nature of the nano-particles themselves, including carbonaceous, metallic, inorganic, and organopolymer based materials.

  10. Thermal and hydrothermal treatment of silica gels as solid stationary phases in gas chromatography.

    PubMed

    El-Naggar, Ashraf Yehia

    2013-01-01

    Silica gel was prepared and treated thermally and hydrothermally and was characterized as solid stationary phase in gas chromatography. The characteristics have been evaluated in terms of polarity, selectivity, and separation efficiencies. These parameters were used to assess the outer silica surface contributions and the degree of surface deactivation brought about by different treatment techniques. The parent silica elutes the paraffinic hydrocarbons with high efficiency of separation and elutes aromatic hydrocarbons with nearly good separation and has bad separation of alcohols. The calcined silica at 500°C and 1000°C has a pronounced effect on the separation of aromatic hydrocarbons compared with the parent silica and hydrothermal treatment of silica. With respect to alcohols separation, the obtained bad separations using treated and untreated silica reflect the little effect of the thermal and hydrothermal treatment on the silica surface deactivation.

  11. Regenerated silica gel as stationary phase on vacuum column chromatography to purify temulawak's extracts

    NASA Astrophysics Data System (ADS)

    Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat, Suzery, Meiny

    2015-12-01

    Commercial silica gel only used once by many researchers and affected high cost for purification process, also less support the green chemistry program. This research focused in regeneration silica gel that used purification of temulawak's extracts (Curcuma xanthorrhiza Roxb) by vacuum column chromatography. Sample extracts (contains 10.1195±0.5971% of curcuminoids) was purified by vacuum column chromatography (pressure: 45 kPa, column: 100mm on length and 16mm on diameter). Ethanol 96% and acetone were compared as eluent. The amount of solvent and yield of curcuminoids used as indicator purification. The silica gel was regenerated with heating in 600°C for 8 hours The silica gels were analyzed by IR spectroscopy and X-ray diffraction. Furthermore, regenerated silica gel was used as the stationary phase in vacuum column chromatography under the same conditions with the previous purification. All the purification experiments were performed in three repetitions. Based on regression equation, y=0.132x+0.0011 (r2=0.9997) the yield of curcuminoids on purified products using ethanol as the eluent was improved 4.26% (to 14.3724±0.5749%) and by acetone was improved 3,03% (to 13.1450 ±0.6318%). The IR spectrum of both silica gel showed the same vibration profile and also there were three crystallinity peaks missing on its X-ray diffraction. Regenerated silica gel has the same performance with new silica gel in purification of temulawak's extract: by ethanol has increased 4.08% (14.1947±0.7415%) and 2.93% (13.0447±0.4822) by acetone. In addition, all purification products showed similar TLC profiles. Purification using regenerated silica gel as the adsorbent on vacuum column chromatography has exactly same potential with the new silica gel.

  12. Regenerated silica gel as stationary phase on vacuum column chromatography to purify temulawak’s extracts

    SciTech Connect

    Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat,; Suzery, Meiny

    2015-12-29

    Commercial silica gel only used once by many researchers and affected high cost for purification process, also less support the green chemistry program. This research focused in regeneration silica gel that used purification of temulawak’s extracts (Curcuma xanthorrhiza Roxb) by vacuum column chromatography. Sample extracts (contains 10.1195±0.5971% of curcuminoids) was purified by vacuum column chromatography (pressure: 45 kPa, column: 100mm on length and 16mm on diameter). Ethanol 96% and acetone were compared as eluent. The amount of solvent and yield of curcuminoids used as indicator purification. The silica gel was regenerated with heating in 600°C for 8 hours The silica gels were analyzed by IR spectroscopy and X-ray diffraction. Furthermore, regenerated silica gel was used as the stationary phase in vacuum column chromatography under the same conditions with the previous purification. All the purification experiments were performed in three repetitions. Based on regression equation, y=0.132x+0.0011 (r{sup 2}=0.9997) the yield of curcuminoids on purified products using ethanol as the eluent was improved 4.26% (to 14.3724±0.5749%) and by acetone was improved 3,03% (to 13.1450 ±0.6318%). The IR spectrum of both silica gel showed the same vibration profile and also there were three crystallinity peaks missing on its X-ray diffraction. Regenerated silica gel has the same performance with new silica gel in purification of temulawak’s extract: by ethanol has increased 4.08% (14.1947±0.7415%) and 2.93% (13.0447±0.4822) by acetone. In addition, all purification products showed similar TLC profiles. Purification using regenerated silica gel as the adsorbent on vacuum column chromatography has exactly same potential with the new silica gel.

  13. Using the liquid nature of the stationary phase in countercurrent chromatography. IV. The cocurrent CCC method.

    PubMed

    Berthod, Alain; Hassoun, Mahmoud

    2006-05-26

    The retention volumes of solutes in countercurrent chromatography (CCC) are directly proportional to their distribution coefficients, K(D) in the biphasic liquid system used as mobile and stationary phase in the CCC column. The cocurrent CCC method consists in putting the liquid "stationary" phase in slow motion in the same direction as the mobile phase. A mixture of five steroid compounds of widely differing polarities was used as a test mixture to evaluate the capabilities of the method with the biphasic liquid system made of water/methanol/ethyl acetate/heptane 6/5/6/5 (v/v) and a 53 mL CCC column of the coil planet centrifuge type. It is shown that the chromatographic resolution obtained in cocurrent CCC is very good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. Pushing the method at its limits, it is demonstrated that the five steroids can still be (partly) separated when the flow rate of the two liquid phases is the same (2 mL/min). This is due to the higher volume of upper phase (72% of the column volume) contained inside the CCC column producing a lower linear speed compared to the aqueous lower phase linear speed. The capabilities of the cocurrent CCC method compare well with those of the gradient elution method in HPLC. Continuous detection is a problem due to the fact that two immiscible liquid phases elute from the column. It was partly solved using an evaporative light scattering detector.

  14. Molecular imprint polymers as highly selective stationary phases for open tubular liquid chromatography and capillary electrochromatography.

    PubMed

    Tan, Z J; Remcho, V T

    1998-09-01

    Chiral separations employing molecular imprint polymer (MIP) stationary phases in both open tubular liquid chromatography (OT-LC) and capillary electrochromatography (OT-CEC) are demonstrated. MIPs are highly crosslinked polymers containing spatial and functionality memory of template molecules which provide a higher degree of selectivity when used as stationary phases for chromatographic separations. Thin films of molecular imprinted polymers bonded to the inner walls of 25 microm ID fused-silica capillaries were prepared using an in situ polymerization technique developed in our laboratory that allows the use of conventional fused-silica capillaries with polyimide outer coatings. The success rate in preparing such open tubular columns was about 70%. Methacrylic acid and 2-vinyl pyridine were chosen as functional monomers, and either ethylene dimethacrylate or trimethylol propane trimethacrylate was used as the crosslinker. Toluene was employed as the porogen. Effects of polymerization conditions on column preparation and chromatographic performance were studied. Enantiomeric separations of D- and L-dansyl phenylalanines were achieved in both OT-LC and OT-CEC modes with good selectivity and efficiencies. Both types of separations may be performed on the same column using a single commercial instrument.

  15. Properties of water as a novel stationary phase in capillary gas chromatography.

    PubMed

    Gallant, Jonathan A; Thurbide, Kevin B

    2014-09-12

    A novel method of separation that uses water as a stationary phase in capillary gas chromatography (GC) is presented. Through applying a water phase to the interior walls of a stainless steel capillary, good separations were obtained for a large variety of analytes in this format. It was found that carrier gas humidification and backpressure were key factors in promoting stable operation over time at various temperatures. For example, with these measures in place, the retention time of an acetone test analyte was found to reduce by only 44s after 100min of operation at a column temperature of 100°C. In terms of efficiency, under optimum conditions the method produced about 20,000 plates for an acetone test analyte on a 250μm i.d.×30m column. Overall, retention on the stationary phase generally increased with analyte water solubility and polarity, but was relatively little correlated with analyte volatility. Conversely, non-polar analytes were essentially unretained in the system. These features were applied to the direct analysis of different polar analytes in both aqueous and organic samples. Results suggest that this approach could provide an interesting alternative tool in capillary GC separations.

  16. Stationary phase modulation in liquid chromatography through the serial coupling of columns: A review.

    PubMed

    Alvarez-Segura, T; Torres-Lapasió, J R; Ortiz-Bolsico, C; García-Alvarez-Coque, M C

    2016-06-07

    Liquid chromatography with single columns often does not succeed in the analysis of complex samples, in terms of resolution and analysis time. A relatively simple solution to enhance chromatographic resolution is the modulation of the stationary phase through the serial coupling of columns. This can be implemented with any type of column using compatible elution conditions and conventional instruments. This review describes the key features of column coupling and published procedures, where two or more columns were coupled in series to solve separation problems. In all reports, the authors could not resolve their samples with single columns, whereas significant enhancement in chromatographic performance was obtained when the columns were combined. Particularly interesting is the reduction in the analysis time in the isocratic mode, which alleviates the "general elution problem" of liquid chromatography, and may represent a stimulus for the proposal of new procedures, especially in combination with mass spectrometric, electrochemical and refractometric detection. Developments proposed to make the serial coupling of columns useful in routine and research laboratories are outlined, including optimisation strategies that facilitate the selection of the appropriate column combination and elution conditions (solvent content, flow rate or temperature) in both isocratic and gradient modes. The availability of zero dead volume couplers, able to connect standard columns, and the commercialisation of short columns with multiple lengths, have expanded the possibilities of success.

  17. Preparation and characterization of six calixarene bonded stationary phases for high performance liquid chromatography.

    PubMed

    Ding, Chenghua; Qu, Kang; Li, Yongbo; Hu, Kai; Liu, Hongxia; Ye, Baoxian; Wu, Yangjie; Zhang, Shusheng

    2007-11-02

    Six calixarene bonded silica gel stationary phases were prepared and characterized by elemental analysis, infrared spectroscopy and thermal analysis. Their chromatographic performance was investigated by using PAHs, aromatic positional isomers and E- and Z-ethyl 3-(4-acetylphenyl) acrylate isomers as probes. Separation mechanism based on the different interactions between calixarenes and analytes were discussed. The chromatographic behaviors of those analytes on the calixarene columns were influenced by the supramolecular interaction including pi-pi interaction, space steric hindrance and hydrogen bonding interaction between calixarenes and analytes. Notably, the presence of polar groups (-OH, -NO(2) and -NH(2)) in the aromatic isomers could improve their separation selectivity on calixarene phase columns. The results from quantum chemistry calculation using DFT-B3LYP/STO-3G* base group were consistent with the retention behaviors of PHAs on calix[4]arene column.

  18. Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations.

    PubMed

    Wimalasinghe, Rasangi M; Breitbach, Zachary S; Lee, Jauh T; Armstrong, Daniel W

    2017-03-01

    Macrocyclic glycopeptide based liquid chromatography stationary phases are known for their highly selective peptide separations. Fast and ultrafast (t R < 1 min) high-efficiency separations were achieved with superficially porous particle (SPP)-based stationary phases. Separations of pharmaceutically important classes of peptides such as enkephalins and bradykinins have been achieved in less than 5 min in isocratic elution modes. Selectivity for peptides structurally similar to one another was increased with use of teicoplanin-based stationary phases compared with commercial C18 stationary phases. Ultrafast isocratic separations of structurally related peptides were achieved with teicoplanin- and vancomycin-based short SPP columns. Acidic mobile phases produced better separations. Ammonium formate was the optimal mobile phase buffer additive. Use of an appropriate combination of a macrocyclic glycopeptide stationary phase and a mobile phase permits faster and more electrospray ionization mass spectrometry compatible isocratic separations than previous gradient approaches. The tryptic peptide separation characteristics of the teicoplanin stationary phase are demonstrated. Additionally, compared with commercial C18 stationary phases, teicoplanin showed tryptic peptide separations with different selectivities. Graphical Abstract Ultrafast separation of enkephalin peptide epimers.

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

    PubMed

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

    2016-05-13

    Stationary phase gradients for chelation thin layer chromatography (TLC) have been investigated as a tool to separate a mixture of metal ions. The gradient stationary phases were prepared using controlled rate infusion (CRI) from precursors containing mono-, bi-, and tri-dentate ligands, specifically 3-aminopropyltriethoxysilane, N-[3-(trimethoxysilyl)propyl] ethylenediamine, and N-[3-(trimethoxysilyl)propyl] diethylenetriamine. The presence and the extent of gradient formation were confirmed using N1s X-ray photoelectron spectroscopy (XPS). XPS results showed that the degree of modification was dependent on the aminosilane precursor, its concentration, and the rate of infusion. The separation of four transition and heavy metals (Co(2+), Pb(2+), Cu(2+), and Fe(3+)) on gradient and uniformly modified plates was compared using a mobile phase containing a stronger chelating agent, ethylenediaminetetraacetic acid (EDTA). The retention of the metal ions was manipulated by varying the surface concentration of the chelating ligands. The order of retention on unmodified plates and on plates modified with a monodentate ligand was Fe(3+)>Cu(2+)∼Pb(2+)∼Co(2+), while the order of retention on plates modified with bi- and tri-dentate ligands was Fe(3+)>Cu(2+)>Pb(2+)∼Co(2+). Fe(3+) and Cu(2+) were much more sensitive to the concentration of chelating ligand on the surface (displaying lower Rf values with increasing ligand concentration) than Pb(2+) and Co(2+). Complete separation was achieved using a high concentration of the tridentate ligand coupled with a longer time for modification, yielding a retention order of Fe(3+)>Cu(2+)>Co(2+)>Pb(2+). Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Development of a V-shape bis(tetraoxacalix[2]arene[2]triazine) stationary phase for high performance liquid chromatography.

    PubMed

    Hu, Kai; Feng, Suxiang; Wu, Mingxia; Wang, Shuang; Zhao, Wenjie; Jiang, Qiong; Yu, Ajuan; Zhang, Shusheng

    2014-12-01

    A new stationary phase for high-performance liquid chromatography was prepared by covalently bonding a V-shape cage heteroatom-bridged calixarene onto silica gel using 3-aminopropyltriethoxysilane as coupling reagent. The structure of the new material was characterized by infrared spectroscopy, elemental analysis and thermogravimetric analysis. Linear solvation energy relationship method was successfully employed to evaluate the new phase with a set of 34 solutes. The retention characteristic of the new phase shows significant similarities with ODS, as well as distinctive features. Multiple mechanisms including hydrophobic, hydrogen bonding, π-π and n-π interaction are involved. The chromatographic behavior of the phase was illustrated by using alkylbenzenes, aromatics positional isomers and flavonoids as probes. Moreover, inorganic anions were individually separated in anion-exchange mode by using the same column. Thus, multi-interaction mechanisms and mixed-mode separation of the new phase can very likely guarantee its excellent chromatographic performance for the analysis of complex samples. The column has been successfully employed for the analysis of clenbuterol in animal urine, and it is demonstrated to be suitable and a competitive alternative analytical method for the determination of clenbuterol.

  1. Fundamental and practical studies on high-performance liquid affinity chromatography of biopolymers with novel stationary phases

    SciTech Connect

    Bacolod, M.D.

    1992-01-01

    Rigid microparticulate stationary phases having surface-bound metal chelating functions were developed and evaluated in high performance metal chelate affinity chromatography of proteins. Silica- and polystyrene-divinylbenzene-based metal chelate sorbents were produced in wide pore and in non-porous type of column packings. A major effort has been placed on development of non-porous highly crosslinked polystyrene-divinylbenzene (PSDVB). These PSDVB microparticles were produced by a two-step swelling polymerization, and exhibited excellent mechanical strength over a wide range of flow-rates and composition used in high performance liquid chromatography (HPLC). Simple and reproducible hydrophilic coatings were developed for the surface modification of hydrophobic PSDVB supports. A tetradentate metal chelating ligand, ethylenediamine-N, N[prime]-diacetic acid (EDDA), was covalently bound to the surface of the various supports. Sorbents having iminodiacetic acid (IDA) metal chelating functions were also evaluated. The hydrophilic character and surface coverage of various stationary phases were assessed chromatographically. Studies concerning the effects of eluent pH as well as the nature and concentration of salts on retention and selectivity with different metal chelate stationary phases having various immobilized metal ions were carried out. Elution schemes were developed for rapid separation of proteins in metal chelate affinity chromatography. EDDA stationary phases in metal forms can be viewed as complementary to IDA stationary phases since they afforded different selectivity and retentivity toward proteins. Hydrophilic PSDVB could be functionalized with IDA or EDDA metal chelating ligands or lectins. The non-porous metal chelate stationary phases afforded rapid separation of proteins by the development of multiple gradient systems, which permitted higher column peak capacity, enabling the separation of a greater number of proteins in a single chromatographic run.

  2. Characterization of the properties of stationary phases for liquid chromatography in aqueous mobile phases using aromatic sulphonic acids as the test compounds.

    PubMed

    Jandera, P; Bocian, S; Molíková, M; Buszewski, B

    2009-01-09

    We investigated the effects of the concentration of naphthalene sulphonic acids (NSAs) as anionic test compounds in the injected sample and of the salt additives to the mobile phase on ion-exclusion. The retention behaviour of NSAs sensitively reflects even minor changes in the ionic and hydrophobic interactions and can be useful for predicting the effects of the stationary phases in reversed-phase chromatography of polar and ionic compounds, both small ones and biopolymers, e.g., oligonucleotides. We studied chromatographic properties of several stationary phases intended for separations in aqueous mobile phases: a C18 column end-capped with polar hydrophilic groups, a densely bonded C8 column doubly end-capped with short alkyl groups, a short alkyl stationary phase designed to keep full pore accessibility in highly-aqueous mobile phases and a Bidentate column with "bridged" C18 groups attached to the silica hydride support. The chemistry and pore structure of various types of column packing materials and of the salt additives to the mobile phase affect the proportion of the pore volume non-accessible to anions due to ion-exclusion and consequently the peak asymmetry and hydrophobic selectivity in reversed-phase chromatography of organic acids. We also addressed the problems connected with the determination of column hold-up volume in aqueous mobile phases. The accessibility of the stationary phase for anionic compounds in contact with the sample zone is affected by ion-exclusion due to repulsive interactions with the negatively charged surface in the pores of the stationary phase. The accessible part of the stationary phase increases and consequently the migration velocity along the column decreases with increasing concentration of the sample in the zone moving along the column. Because of a limited access to the stationary phase, its capacity can be easily overloaded. The combination of the column overload and ion-exclusion effects may result in fronting or

  3. Retention behavior on aminoethyl-modified poly(p-phenylene terephthalamide) fiber stationary phases in gas chromatography.

    PubMed

    Shirai, Shingoro; Saito, Yoshihiro; Sakurai, Yasuhiro; Ueta, Ikuo; Jinno, Kiyokatsu

    2010-01-01

    Surface derivatization of Kevlar, poly(p-phenylene terephthalamide), fiber has been studied along with the evaluation of the surface characteristics of the chemically-modified fiber as the stationary phase in packed-capillary gas chromatography (GC). Several experimental parameters in the derivatization reaction have been optimized, and the retention behavior of the surface-derivatized fibrous stationary phase has been investigated using various standard solutes, such as alkanes, alcohols and alkylbenzenes. By introducing aminoethyl functional groups onto the surface of the fibrous material, a specific selectivity for polar solutes has been observed.

  4. A new anion-exchange/hydrophobic monolith as stationary phase for nano liquid chromatography of small organic molecules and inorganic anions.

    PubMed

    Aydoğan, Cemil

    2015-05-01

    In this study, an anion-exchange/hydrophobic polymethacrylate-based stationary phase was prepared for nano-liquid chromatography of small organic molecules and inorganic anions. The stationary phase was synthesized by in situ polymerization of 3-chloro-2-hydroxypropylmethacrylate and ethylene dimethacrylate inside silanized 100 μm i.d. fused silica capillary. The porogen mixture consisted of toluene and dodecanol. The pore size distrubution profiles of the resulting monolith were determined by mercury intrusion porosimetry and the morphology of the prepared monolith was investigated by scanning electron microscope. Good permeability, stability and column efficiency were observed on the monolithic column with nano flow. The produced monolithic column, which contains reactive chloro groups, was then modified by reaction with N,N-dimethyl-N-dodecylamine to obtain an anion-exchange/hydrophobic monolithic stationary phase. The functionalized monolith contained ionizable amine groups and hydrophobic groups that are useful of anion-exchange/hydrophobic mixed-mode chromatography. The final monolithic column performance with respect to anion-exchange and hydrophobic interactions was assesed by the separation of alkylbenzene derivatives, phenolic compounds and inorganic anions, respectively. Theoretical plate numbers up to 23,000 plates/m were successfully achieved in the separation of inorganic anions.

  5. Separation of parabens on a zirconia-based stationary phase in superheated water chromatography.

    PubMed

    Yarita, Takashi; Aoyagi, Yoshie; Sasai, Haruka; Nishigaki, Atsuko; Shibukawa, Masami

    2013-01-01

    A superheated water chromatography (SWC) method for the separation of alkyl esters of 4-hydroxybenzoic acid (parabens) using a zirconia-based stationary phase was developed and applied to real sample analysis. First, the SWC system was optimized in terms of the proper length of the preheating coil for establishing thermal equilibration of the mobile phase entering the column at the oven temperature. Next, the effect of the column temperature on the retention was investigated at 100-180°C. The elution time for all parabens decreased with increasing column temperature, and linear relationships between ln k and 1/T were obtained. At higher column temperatures, the elution time was further shortened because of the increased mobile-phase flow rate. Nevertheless, the loss of column efficiency at the higher flow rates was not significant. The application of the present method to the analysis of commercial lotions was then demonstrated. The quantification results obtained from SWC showed good agreement with those from a conventional HPLC method.

  6. From analytical methods to large scale chiral supercritical fluid chromatography using chlorinated chiral stationary phases.

    PubMed

    Wu, Dauh-Rurng; Yip, Shiuhang Henry; Li, Peng; Sun, Dawn; Mathur, Arvind

    2016-02-05

    While traditional non-chlorinated Cellulose- and Amylose-derivatized phases have been used successfully in supercritical fluid chromatography (SFC) to resolve a broad variety of chiral compounds, some chiral pharmaceutical compounds are not well resolved on these traditional chiral stationary phases (CSP) due to the lack of chiral selectivity. Since there are no universal CSP to resolve all chiral compounds, chlorinated CSP can be complementary to the non-chlorinated CSP. Chlorinated CSP such as 4-Chloro-3-methylphenyl-carbamatecellulose (Lux-Cellulose-4), 3-Chloro-4-methylphenyl-carbamatecellulose (Lux-Cellulose-2), 5-Chloro-2-methylphenyl-carbamateamylose (Lux-Amylose-2) and immobilized 3,5-dichlorophenyl-carbamatecellulose (Chiralpak IC) have provided a range of chiral recognition mechanisms which have allowed the authors to successfully achieve chiral SFC resolution on several structurally diverse compounds, which are not well resolved in the non-chlorinated CSP. In addition, chlorinated Lux-Cellulose-4, Chiralpak IC and Lux-Amylose-2 have enabled us to utilize non-alcohol solvents as sample diluents and as co-solvents to significantly improve compound solubility and selectivity. This article will discuss the challenges associated with several SFC applications on both coated and immobilized chlorinated CSP to deliver high-quality drug candidates in large quantity. The use of dichloromethane in both sample preparation and as co-solvent in CO2 to increase sample solubility will be presented in preparative example #2 and #3.

  7. New chiral stationary phases based on xanthone derivatives for liquid chromatography.

    PubMed

    Fernandes, Carla; Tiritan, Maria Elizabeth; Cravo, Sara; Phyo, Ye' Zaw; Kijjoa, Anake; Silva, Artur M S; Cass, Quezia B; Pinto, Madalena M M

    2017-08-01

    Six chiral derivatives of xanthones (CDXs) were covalently bonded to silica, yielding the corresponding xanthonic chiral stationary phases (XCSPs). The new XCSPs were packed into stainless-steel columns with 150 x 4.6 mm i.d. Moreover, the greening of the chromatographic analysis by reducing the internal diameter (150 x 2.1 mm i.d.) of the liquid chromatography (LC) columns was also investigated. The enantioselective capability of these phases was evaluated by LC using different chemical classes of chiral compounds, including several types of drugs. A library of CDXs was evaluated in order to explore the principle of reciprocity as well as the chiral self-recognition phenomenon. The separation of enantiomeric mixtures of CDXs was investigated under multimodal elution conditions. The XCSPs provided high specificity for the enantiomeric mixtures of CDXs evaluated mainly under normal-phase elution conditions. Furthermore, two XCSPs were prepared with both enantiomers of the same xanthonic selector in order to confirm the inversion order elution. © 2017 Wiley Periodicals, Inc.

  8. Suitability of silica hydride stationary phase, aqueous normal phase chromatography for untargeted metabolomic profiling of Enterococcus faecium and Staphylococcus aureus.

    PubMed

    Weisenberg, Scott A; Butterfield, Tiffany R; Fischer, Steven M; Rhee, Kyu Y

    2009-07-01

    We report the robustness of silica hydride stationary phase, aqueous normal phase (ANP) chromatography to the chemical complexity of the intracellular metabolomes of Staphylococcus aureus and Enterococcus faecium. We specifically demonstrate that the chromatographic behavior of known metabolites is unaffected by the intracellular chemical matrix of these microbes and that this method enables untargeted profiling of their intracellular metabolites using accurate mass-retention time (AMRT) identifiers. We further demonstrate the ability of AMRT-based metabolite profiling to differentiate bacteria along genetic and phenotypic lines. Overall, these data commend the utility of ANP-based chromatography for untargeted metabolomics-based studies of microbial physiology and antibiotic resistance.

  9. [Separation of purines, pyrimidines, pterins and flavonoids on magnolol-bonded silica gel stationary phase by high performance liquid chromatography].

    PubMed

    Chen, Hong; Li, Laishen; Zhang, Yang; Zhou, Rendan

    2012-10-01

    A new magnolol-bonded silica gel stationary phase (MSP) was used to separate the basic drugs including four purines, eight pyrimidines, four pterins and five flavonoids as polar representative samples by high performance liquid chromatography (HPLC). To clarify the separation mechanism, a commercial ODS column was also tested under the same chromatographic conditions. The high selectivities and fast baseline separations of the above drugs were achieved by using simple mobile phases on MSP. Although there is no end-caped treatment, the peak shapes of basic drugs containing nitrogen such as purines, pyrimidines and pterins were rather symmetrical on MSP, which indicated the the magnolol as ligand with multi-sites could shield the side effect of residual silanol groups on the surface of silica gel. Although somewhat different in the separation resolution, it was found that the elution orders of some drugs were generally similar on both MSP and ODS. The hydrophobic interaction should play a significant role in the separations of the above basic drugs, which was attributed to their reversed-phase property in the nature. However, MSP could provide the additional sites for many polar solutes, which was a rational explanation for the high selectivity of MSP. For example, in the separation of purines, pyrimidines and pterins on MSP, hydrogen-bonding and dipole-dipole interactions played leading roles besides hydrophobic interaction. Some solute molecules (such as mercaptopurine, vitexicarpin) and MSP can form the strong pi-pi stacking in the separation process. All enhanced the retention and improved the separation selectivity of MSP, which facilitated the separation of the basic drugs.

  10. Spherical ordered mesoporous silicas and silica monoliths as stationary phases for liquid chromatography.

    PubMed

    Galarneau, Anne; Iapichella, Julien; Brunel, Daniel; Fajula, François; Bayram-Hahn, Zöfre; Unger, Klaus; Puy, Guillaume; Demesmay, Claire; Rocca, Jean-Louis

    2006-04-01

    Ordered mesoporous silicas such as micelle-templated silicas (MTS) feature unique textural properties in addition to their high surface area (approximately 1000 m2/g): narrow mesopore size distributions and controlled pore connectivity. These characteristics are highly relevant to chromatographic applications for resistance to mass transfer, which has never been studied in chromatography because of the absence of model materials such as MTS. Their synthesis is based on unique self-assembly processes between surfactants and silica. In order to take advantage of the perfectly adjustable texture of MTS in chromatographic applications, their particle morphology has to be tailored at the micrometer scale. We developed a synthesis strategy to control the particle morphology of MTS using the concept of pseudomorphic transformation. Pseudomorphism was recognized in the mineral world to gain a mineral that presents a morphology not related to its crystallographic symmetry group. Pseudomorphic transformations have been applied to amorphous spherical silica particles usually used in chromatography as stationary phases to produce MTS with the same morphology, using alkaline solution to dissolve progressively and locally silica and reprecipitate it around surfactant micelles into ordered MTS structures. Spherical beads of MTS with hexagonal and cubic symmetries have been synthesized and successfully used in HPLC in fast separation processes. MTS with a highly connected structure (cubic symmetry), uniform pores with a diameter larger than 6 nm in the form of particles of 5 microm could compete with monolithic silica columns. Monolithic columns are receiving strong interest and represent a milestone in the area of fast separation. Their synthesis is a sol-gel process based on phase separation between silica and water, which is assisted by the presence of polymers. The control of the synthesis of monolithic silica has been systematically explored. Because of unresolved yet

  11. Latex-coated polymeric monolithic ion-exchange stationary phases. 2. Micro-ion chromatography.

    PubMed

    Zakaria, Philip; Hutchinson, Joseph P; Avdalovic, Nebojsa; Liu, Yan; Haddad, Paul R

    2005-01-15

    Latex-coated monolithic polymeric stationary phases are used for micro-ion chromatography (mu-IC) of inorganic anions. Monolithic columns were prepared by the in situ polymerization of butyl methacrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid within fused-silica capillaries of varying internal diameters. Introduction of ion-exchange sites was achieved by coating the anionic polymeric monolith with either Dionex AS10 or Dionex AS18 quaternary ammonium functionalized latex particles to give total ion-exchange capacities in the range 9-24 nequiv for a 30-cm column. The resultant mu-IC columns were used for the separation of anionic analytes using chloride or acetate as the eluent-competing ion and direct UV spectrophotometric detection at 195 nm or using hydroxide as the eluent-competing ion and suppressed or nonsuppressed contactless conductivity detection. Separation efficiencies of 13,000 plates/m were observed (for iodate), and separation efficiency was maintained for large increases in flow rate (up to 42 microL/min, corresponding to a linear flow velocity of 18.5 mm/s), enabling highly reproducible, rapid separations to be achieved (seven analyte anions in less than 2 min). Use of a hollow fiber micromembrane suppressor enabled effective suppression of hydroxide eluents over the range 0.5-5.0 mM, thereby permitting suppressed conductivity detection to be performed. However, the relatively large size of the suppressor resulted in reduced separation efficiencies (e.g., 5400 plates/m for iodate). Detection limits obtained with suppressed conductivity detection were in the range 0.4-1.2 microM.

  12. Chiral stationary phases based on chitosan bis(methylphenylcarbamate)-(isobutyrylamide) for high-performance liquid chromatography.

    PubMed

    Tang, Sheng; Bin, Qin; Chen, Wei; Bai, Zheng-Wu; Huang, Shao-Hua

    2016-04-01

    A series of chitosan bis(methylphenylcarbamate)-(isobutyrylamide) derivatives were synthesized by carbamylating chitosan isobutyrylamide with different methylphenyl isocyanates. Then the prepared chitosan derivatives were coated onto 3-aminopropyl silica particles, resulting in a series of new chiral stationary phases (CSPs) for high-performance liquid chromatography. It was observed that the chiral recognition abilities of these coated-type CSPs depended very much on the substituents on the phenyl moieties of the chitosan derivatives, the eluent composition, as well as the structure of racemates. As a typical example, the eluent tolerance of the prepared CSP with the best enantioseparation ability was investigated in detail, and the results revealed that the CSP exhibited extraordinary solvent tolerance and could still work without significant loss in enantioseparation capability after being flushed with chloroform (100%), ethyl acetate (100%) and even THF/n-hexane (70/30, v/v), while the traditional coated-type CSPs based on the cellulose and amylose derivatives, such as cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) and amylose tris(3,5-dimethylphenylcarbamate) (ADMPC), might be dissolved or highly swollen in these eluents. Therefore, the application of the resultant CSPs could address the problem of the dissolution and high swelling of traditional coated-type CSPs in some unusual eluents, broadening the possibility of eluent choice. In addition, a comparison of the prepared CSPs with the well known CDMPC- and ADMPC- based CSPs concerning the chiral recognition ability was also made. Separation performances achieved on the as-prepared CSPs in different eluents were found to be even superior to CDMPC- and ADMPC-based CSPs for the tested chiral compounds. In summary, we could safely draw the conclusion that the CSPs derived from chitosan isobutyrylamide derivatives were capable of excellent chiral recognition ability, and meanwhile possessed satisfactory

  13. A mechanistic model of ion-exchange chromatography on polymer fiber stationary phases.

    PubMed

    Winderl, Johannes; Hahn, Tobias; Hubbuch, Jürgen

    2016-12-02

    Fibers are prominent among novel stationary phase supports for preparative chromatography. Several recent studies have highlighted the potential of fiber-based adsorbents for high productivity downstream processing in both batch and continuous mode, but so far the development of these materials and of processes employing these materials has solely been based on experimental data. In this study we assessed whether mechanistic modeling can be performed on fiber-based adsorbents. With a column randomly filled with short cut hydrogel grafted anion exchange fibers, we tested whether tracer, linear gradient elution, and breakthrough data could be reproduced by mechanistic models. Successful modeling was achieved for all of the considered experiments, for both non-retained and retained molecules. For the fibers used in this study the best results were obtained with a transport-dispersive model in combination with a steric mass action isotherm. This approach accurately accounted for the convection and dispersion of non-retained tracers, and the breakthrough and elution behaviors of three different proteins with sizes ranging from 6 to 160kDa were accurately modeled, with simulation results closely resembling the experimental data. The estimated model parameters were plausible both from their physical meaning, and from an analysis of the underlying model assumptions. Parameters were determined within good confidence levels; the average confidence estimate was below 7% for confidence levels of 95%. This shows that fiber-based adsorbents can be modeled mechanistically, which will be valuable for the future design and evaluation of these novel materials and for the development of processes employing such materials.

  14. Enantiodifferentiation of whisky and cognac lactones using gas chromatography with different cyclodextrin chiral stationary phases.

    PubMed

    Schmarr, Hans-Georg; Mathes, Maximilian; Wall, Kristina; Metzner, Frank; Fraefel, Marius

    2017-09-22

    The chiral lactone 5-butyl-4-methyloxolan-2-one or 5-butyl-4-methyldihydro-2(3H)-furanone, often named whisky lactone, is found in oak wood, then contributing to the appreciated flavor of beverages stored in such wooden barrels. Its next higher homologue is named cognac lactone (5-pentyl-4-methyloxolan-2-one or 5-pentyl-4-methyldihydro-2(3H)-furanone), however is much less known, probably due to its minor concentration level. In order to study the direct enantioseparation of both lactones by gas chromatography on chiral stationary phases, individual enantiomers, particularly for cognac lactone were made available. This was achieved by baker's yeast reduction of synthesized ethyl 3-methyl-4-oxononanoate or, after hydrolysis, of the corresponding 4-ketoacid, that gave access to individual enantiomers of cognac lactone. Good enantioseparation was achieved for both whisky and cognac lactone with high values for the chiral resolution with 6-O-tert. butyl dimethylsilyl-2,3-dialkylated or 6-O-tert. butyl dimethylsilyl-2,3-diacylated cyclodextrin derivatives as chiral selectors. The influence of the nature and position of derivatization of the cyclodextrin moiety revealed a strong impact on the chiral recognition mechanism, as the investigated alkylated derivatives heptakis-(2,6-di-O-iso-pentyl-3-O-allyl)-β-cyclodextrin and octakis-(2,3-di-O-pentyl-6-O-methyl)-γ-cyclodextrin did not provide any or only minor chiral selectivity for the two lactones. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Retention behavior of pyridinium oximes on PFP stationary phase in high-performance liquid chromatography.

    PubMed

    Dohnal, Vlastimil; Musílek, Kamil; Kuča, Kamil

    2014-03-01

    The chromatographic behavior was studied of a series of potential acetylcholinesterase reactivators, pyridinium oximes, bearing linear aliphatic chains of the length of the aliphatic bridge from 1 to 12 carbon atoms, on a pentafluorophenyl-modified stationary phase. The retention mechanisms and the dependence of the capacity factor on mobile phase composition, aliphatic chain bridge length and calculated log P were evaluated and discussed in detail. The separation of the studied oximes was found to be driven by hydrophobic interactions when a lower content of organic modifier was used in mobile phase; however, the ion-exchange mechanism was the leading one when a large portion of organic modifier was used. In addition, the lipophilicity was found to be a driving mechanism of the separation of oximes bearing a connecting chain of the length of 6-12 carbon atoms, whereas the retention of oximes with shorter connecting chains was significantly influenced by other separation mechanisms such as aromatic or π-π interaction. These results can be useful for the development of new, efficient acetylcholinesterase reactivators.

  16. Polar stationary phases based on poly(oligo ethylene glycol)diacrylates for capillary gas chromatography

    NASA Astrophysics Data System (ADS)

    Shiryaeva, V. E.; Popova, T. P.; Korolev, A. A.; Kanat'eva, A. Yu.; Kurganov, A. A.

    2017-08-01

    New stationary phases for capillary columns in GC are synthesized and studied. The phases are prepared by depositing oligo(ethylene glycol)diacrylates on the column walls and subsequent polymerization (crosslinking) in the presence of peroxide initiators. It is shown that stationary phases based on monomers with molecular weights of 10 kDa or higher exhibit separation properties similar to those of conventional stationary phases based on polyethylene glycol (PEG); however, their thermal stability is higher because they have a higher degree of crosslinking and a more ordered structure of the crosslinked polymers than the respective parameters of phases based on native PEG.

  17. A micro gas chromatography with separation capability enhanced by polydimethylsiloxane stationary phase functionalized by carbon nanotubes and graphene.

    PubMed

    Li, Yubo; Zhang, Runzhou; Wang, Tao; Wang, Youhao; Wang, Yonghuan; Li, Lingfeng; Zhao, Weijun; Wang, Xiaozhi; Luo, Jikui

    2016-07-01

    Polydimethylsiloxane (PDMS) stationary phases functionalized with multi-walled carbon nanotubes (MWCNTs) and graphene, respectively, for the columns in micro gas chromatography are presented in this paper. To exploit the merits of MWCNTs and graphene in terms of their high specific surface area, low surface energy and chemical inertness, experimental conditions for separation (heating rate and final temperature of temperature programming, flow rate of carrier gas and the volume of samples injection) are investigated, and separations of both polar and nonpolar compound mixtures under these conditions are performed. Compared with PDMS-only coated stationary phases, the functionalization of the phases with carbon nano-materials improves the performance of columns in separation, repeatability, stability and revolution significantly. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. [Preparation and evaluation of 2,6-di-O-pentyl-beta-cyclodextrin bonded silica stationary phase for high performance liquid chromatography].

    PubMed

    Liu, Liwen; Luo, Aiqin; Dai, Rongji; Ge, Xiaoxia; Yang, Shaoning

    2004-11-01

    In order to improve the chiral separation capability of the conventional beta-cyclodextrin bonded-silica gel stationary phase, 2,6-di-O-pentyl-beta-cyclodextrin bonded stationary phase (PCDS) was prepared via a long spacer. The resulted bonded-silica stationary phase was characterized by three methods, namely Fourier transform infrared, Molisch color reaction, X-ray optical electrical energy spectrogram. The chromatographic performances of PCDS were investigated by using liquid chromatography with toluene, dimethyl phthalate, and phenanthrene as solutes, and their retention mechanism was investigated and discussed. The results show that the introduction of pentyl to beta-cyclodextrin leads to enhancement of the retention of the solutes. The chiral separation capability of the new bonded-silica stationary phase was evaluated by using liquid chromatography with some chiral drugs. Some of the enantiomers such as chlorphenamine maleate and bupropion hydrochloride were separated by heptakis (2,6-di-O-pentyl)-beta-cyclodextrin bonded silica stationary.

  19. Lipidic ionic liquid stationary phases for the separation of aliphatic hydrocarbons by comprehensive two-dimensional gas chromatography.

    PubMed

    Nan, He; Zhang, Cheng; O'Brien, Richard A; Benchea, Adela; Davis, James H; Anderson, Jared L

    2017-01-20

    Lipidic ionic liquids (ILs) possessing long alkyl chains as well as low melting points have the potential to provide unique selectivity as well as wide operating ranges when used as stationary phases in gas chromatography. In this study, a total of eleven lipidic ILs containing various structural features (i.e., double bonds, linear thioether chains, and cyclopropanyl groups) were examined as stationary phases in comprehensive two dimensional gas chromatography (GC×GC) for the separation of nonpolar analytes in kerosene. N-alkyl-N'-methyl-imidazolium-based ILs containing different alkyl side chains were used as model structures to investigate the effects of alkyl moieties with different structural features on the selectivities and operating temperature ranges of the IL-based stationary phases. Compared to a homologous series of ILs containing saturated side chains, lipidic ILs exhibit improved selectivity toward the aliphatic hydrocarbons in kerosene. The palmitoleyl IL provided the highest selectivity compared to all other lipidic ILs as well as the commercial SUPELCOWAX 10 column. The linoleyl IL containing two double bonds within the alkyl side chain showed the lowest chromatographic selectivity. The lipidic IL possessing a cyclopropanyl group within the alkyl moiety exhibited the highest thermal stability. The Abraham solvation parameter model was used to evaluate the solvation properties of the lipidic ILs. This study provides the first comprehensive examination into the relation between lipidic IL structure and the resulting solvation characteristics. Furthermore, these results establish a basis for applying lipidic ILs as stationary phases for solute specific separations in GC×GC. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. [Preparation and evaluation of octadecanethiol modified gold microspheres in capillary liquid chromatography and pressurized capillary electrochromatography as stationary phase].

    PubMed

    Zhao, Zhenzhen; Qu, Qishu; Zhang, Xinxin; Gu, Xue; Wang, Yan; Yan, Chao

    2009-07-01

    Gold microspheres modified with octadecanethiol as chromatographic stationary phase were prepared. The particles were characterized by the scanning electron micrograph (SEM), Fourier transform infrared spectroscopy (FT-IR), elemental analysis and nitrogen adsorption analysis. The average diameter, the surface area and the average pore diameter were 3.5 microm, 49.0 m2/g and 5.0 nm, respectively. The IR spectra demonstrated that C18 was bonded to the surface of gold microspheres with the carbon content of 0.56%. Using these microspheres as stationary phase, a 19 cm section of a total length of 36 cm capillary (100 microm i. d.) was packed electrokinetically, and the evaluations in capillary liquid chromatography and pressurized capillary electrochromatography were performed. The mobile phases (80% methanol) with extreme pH values (pH 1.0 or pH 12.0) were used to flush the column for 140 h. In order to investigate the chemical stability of the column, the retention factors before and after flushing were calculated and compared based on the experimental results. There was no remarkable deterioration on the retention factors after flushing, which demonstrated the column was stable pounds were separated using capillary liquid chromatography to examine the retention behavior of the column, and over 50,000 theoretical plates per meter and acceptable symmetry peaks were obtained. The pressurized capillary electrochromatographic properties of the column were investigated using a separation of the mixture of aniline and benzoic acid, and the separation was obtained when a 5 kV positive or negative voltage was applied. The research work confirmed the feasibility of using the octadecanethiol modified gold microspheres as a novel stationary phase for capillary liquid chromatography and pressurized capillary electrochromatography.

  1. Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

    PubMed

    Sonomura, Kazuhiro; Kudoh, Shinobu; Sato, Taka-Aki; Matsuda, Fumihiko

    2015-06-01

    A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono-hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R(2) > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co-existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.

  2. Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

    PubMed

    Periat, Aurélie; Krull, Ira S; Guillarme, Davy

    2015-02-01

    This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Effects of supercritical fluid chromatography conditions on enantioselectivity and performance of polyproline-derived chiral stationary phases.

    PubMed

    Novell, Arnau; Méndez, Alberto; Minguillón, Cristina

    2015-07-17

    The chromatographic behaviour and performance of four polyproline-derived chiral stationary phases (CSPs) were tested using supercritical fluid chromatography (SFC). A series of structurally related racemic compounds, whose enantioseparation was proved to be sensitive to the type of mobile phase used in NP-HPLC, were chosen to be tested in the SFC conditions. Good enantioselection ability was shown by the CSPs for the analytes tested in the new conditions. Resolution, efficiency and analysis time, were considerably improved with respect to NP-HPLC when CO2/alcohol mobile phases were used. Monolithic columns clearly show enhanced chromatographic parameters and improved performance respect to their bead-based counterparts.

  4. On-chip temperature gradient interaction chromatography.

    PubMed

    Shih, Chi-Yuan; Chen, Yang; Xie, Jun; He, Qing; Tai, Yu-Chong

    2006-04-14

    This paper reports the first integrated microelectromechanical system (MEMS) HPLC chip that consists of a parylene high-pressure LC column, an electrochemical sensor, a resistive heater and a thermal-isolation structure for on-chip temperature gradient interaction chromatography application. The separation column was 8 mm long, 100 microm wide, 25 microm high and was packed with 5 microm sized, C18-coated beads using conventional slurry-packing technique. A novel parylene-enhanced, air-gap thermal isolation technology was used to reduce heater power consumption by 58% and to reduce temperature rise in the off-column area by 67%. The fabricated chip consumed 400 mW when operated at 100 degrees C. To test the chromatography performance of the fabricated system, a mixture of derivatized amino acids was chosen for separation. A temporal temperature gradient scanning from 25 to 65 degrees C with a ramping rate of 3.6 degrees C/min was applied to the column during separation. Successful chromatographic separation of derivatized amino acids was carried out using our chip. Compared with conventional temperature gradient HPLC system which incorporates "macro oven" to generate temporal temperature gradient on the column, our chip's thermal performance, i.e., power consumption and thermal response, is greatly improved without sacrificing chromatography quality.

  5. Hydrophilic interaction liquid chromatography in food analysis.

    PubMed

    Bernal, José; Ares, Ana M; Pól, Jaroslav; Wiedmer, Susanne K

    2011-10-21

    The use of hydrophilic interaction liquid chromatography (HILIC) in food analysis in the last decade is reviewed. The HILIC mechanism is briefly discussed, but main emphasis is put on the use of HILIC for separation of different food matrices. The food matrices are divided into food of animal origin and related products, vegetables, fruits and related compounds, and other food-related matrices. A list on important applications is provided for each category including experimental conditions and a brief summary of the results. The 100 references included will provide the reader a comprehensive overview and insight into HILIC applications to food analysis. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Preparation and retention mechanism study of graphene and graphene oxide bonded silica microspheres as stationary phases for high performance liquid chromatography.

    PubMed

    Zhang, Xiaoqiong; Chen, Sha; Han, Qiang; Ding, Mingyu

    2013-09-13

    Graphene oxide (GO) bonded stationary phase for high performance liquid chromatography (HPLC) was fabricated by coating GO sheets onto aminosilica microspheres via covalent coupling. Graphene (G) functionalized HPLC stationary phase was then prepared through hydrazine reduction of GO bonded silica (GO@SiO2) composite, which was the first example of using graphene as stationary-phase component for HPLC. Effective separations of the tested neutral and polar compounds on both GO@SiO2 and graphene bonded silica (G@SiO2) columns were achieved under the optimal experimental conditions. Compared with commercial C18 column, the different chromatographic performances of GO and graphene bonded columns were ascribed to their unique retention mechanisms. The polyaromatic scaffold of GO and graphene gives π-π stacking property and hydrophobic effect, and other retention mechanisms, such as π-π electron-donor-acceptor (EDA) interaction for the separation of nitroaromatic compounds and hydrogen bonding for hydroxyl and amino compounds, may also be taken into consideration. Experimental results indicated that the mixed-mode retention mechanism can facilitate the separation of analytes with similar hydrophobicity, which is a unique property compared with C18 column. Additionally, G@SiO2 showed higher affinity to aromatic analytes in contrast with GO@SiO2 and its retention mechanism was not consistent with the typical reversed phase behavior. The separation of aromatic compounds on G@SiO2 column relies primarily on the π-π stacking interaction and then the hydrophobicity, while the two interactions have equal shares on GO@SiO2 column.

  7. Application of hydrophilic interaction chromatography for simultaneous separation of six impurities of mildronate substance.

    PubMed

    Hmelnickis, Juris; Pugovics, Osvalds; Kazoka, Helena; Viksna, Arturs; Susinskis, Igors; Kokums, Kaspars

    2008-11-04

    The possibility of separating the impurities of mildronate, an antiischemic drug, by hydrophilic interaction chromatography (HILIC) was investigated on different polar stationary phases (silica, amino, cyano and zwitterionic sulfobetaine). The investigations have shown that HILIC is a useful alternative to reversed phase and ion-pair chromatography. The impact of HILIC separation conditions (acetonitrile content, buffer pH in mobile phase) on retention and selectivity has been systematically studied. Importance of these factors was found to be dependent on the structural properties of solutes. A HILIC method using a zwitterionic sulfobetaine stationary phase was developed and validated to determine six impurities in the drug substance. The method was validated in terms of specificity, limit of quantitation, limit of detection, linearity, accuracy and precision.

  8. Silica gel microspheres decorated with covalent triazine-based frameworks as an improved stationary phase for high performance liquid chromatography.

    PubMed

    Zhao, Wenjie; Hu, Kai; Hu, Chenchen; Wang, Xiaoyu; Yu, Ajuan; Zhang, Shusheng

    2017-03-03

    A new stationary phase for high performance liquid chromatography (HPLC) applications based on silica gel microspheres decorated with covalent triazine-based frameworks (CTF-SiO2) composite has been reported here. In this new hybrid material, sheet-like covalent triazine-based frameworks (CTF) were grown onto the supporting silica spheres, in order to achieve improved chromatographic separation and selectivity. The new material was characterized by infrared spectroscopy, elemental analysis and thermogravimetric analysis. The chromatographic performance and retention mechanism of the new stationary phase were investigated in reversed-phase mode and compared against standard C18 and cyano-modified silica (CN-SiO2) columns. A variety of different probe molecules were analyzed, including mono-substituted benzenes, polycyclic aromatic hydrocarbons, phenols, anilines and bases. The synergism of triazine and aromatic moieties provided several different retention mechanisms, thus improving the selectivity in the CTF-SiO2 composite. The good column packing properties of the uniform silica microspheres combined with the separation ability of the CTF frameworks make the new CTF-SiO2 material a potentially useful stationary phase for the analysis of complex samples.

  9. Silica sputtering as a novel collective stationary phase deposition for microelectromechanical system gas chromatography column: feasibility and first separations.

    PubMed

    Vial, J; Thiébaut, D; Marty, F; Guibal, P; Haudebourg, R; Nachef, K; Danaie, K; Bourlon, B

    2011-05-27

    Since the late 1970s, approaches have been proposed to replace conventional gas chromatography apparatus with silicon-based microfabricated separation systems. Performances are expected to be improved with miniaturization owing to the reduction of diffusion distances and better thermal management. However, one of the main challenges consists in the collective and reproducible fabrication of efficient microelectromechanical system (MEMS) gas chromatography (GC) columns. Indeed, usual coating processes or classical packing with particulate matters are not compatible with the requirements of collective MEMS production in clean room facilities. A new strategy based on the rerouting of conventional microfabrication techniques and widely used in electronics for metals and dielectrics deposition is presented. The originality lies in the sputtering techniques employed for the deposition of the stationary phase. The potential of these novel sputtered stationary phases is demonstrated with silica sputtering applied to the separation of light hydrocarbons and natural gases. If kinetic characteristics of the sputtered open tubular columns were acceptable with 2500 theoretical plates per meter, the limited retention and resolution of light hydrocarbons led us to consider semipacked sputtered columns with rectangular pillars allowing also significant reduction of typical diffusion distances. In that case separations were greatly improved because retention increased and efficiency was close to 5000 theoretical plates per meter. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Interaction between Antibacterial Peptide Apep10 and Escherichia coli Membrane Lipids Evaluated Using Liposome as Pseudo-Stationary Phase

    PubMed Central

    Li, Man

    2017-01-01

    Liposomes constructed from Escherichia coli membrane lipids were used as a pseudo-stationary phase in capillary electrophoresis and immobilised liposome chromatography to evaluate the interaction between antibacterial peptide (ABP) Apep10 and bacterial membrane lipids. The peptide mobility decreased as the concentration of liposomes increased, providing evidence for the existence of this interaction. The binding constant between Apep10 and the Escherichia coli membranes lipid liposome was higher than that of Apep10 with a mixed phospholipids liposome at the same temperature. The capillary electrophoresis results indicate that the binding ability of Apep10 with a liposome was dependent on the liposome’s lipid compositions. Thermodynamic analysis by immobilised liposome chromatography indicated that hydrophobic and electrostatic effects contributed to the partitioning of Apep10 in the membrane lipids. The liposomes constructed from bacterial membrane lipid were more suitable as the model membranes used to study dynamic ABP/membrane interactions than those constructed from specific ratios of particular phospholipids, with its more biomimetic phospholipid composition and contents. This study provides an appropriate model for the evaluation of ABP-membrane interactions. PMID:28052090

  11. Hydrophilic interaction chromatography for the analysis of aminoglycosides.

    PubMed

    Kumar, Praveen; Rubies, Antoni; Companyó, Ramon; Centrich, Francesc

    2012-02-01

    The effect of mobile-phase constituents (pH and ionic strength) and chromatographic behaviour of ten aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, apramycin, paramomycin, kanamycin A, gentamycin C1, gentamycin C2/C2a, gentamycin C1a and neomycin) in the bare silica, amino, amide and zwitterionic phases of hydrophilic interaction chromatography (HILIC) were studied systematically. Among the stationary phases studied, the zwitterionic phase provided the best separation of aminoglycosides. The effect of pH, ionic concentration and column temperature on retention time, peak shape and sensitivity was studied using a central composite design. pH affected sensitivity of the detection of analytes but not the retention time. High ionic strength in the mobile phase was necessary to control the ionic interactions between ionised aminoglycosides and the hydrophilic phase, thereby influencing peak shape and retention time. Column temperature affected sensitivity of the detection but not the retention time. During method development, crosstalk between the MS/MS channels of the analytes was observed and resolved.

  12. Hydrophilic interaction liquid chromatography with alcohol as a weak eluent.

    PubMed

    Liu, Min; Ostovic, Judy; Chen, Emily X; Cauchon, Nina

    2009-03-20

    There has been a significant increase of interest in polar compound separation by hydrophilic interaction liquid chromatography (HILIC), in which acetonitrile is mostly used as a weak eluent. Although replacing acetonitrile with alcohols as organic modifiers has been previously reported, the separation mechanism was poorly understood. In this paper we explored the separation mechanism through the method development for the analysis of the trace amounts of polar and basic hydrazines, which were genotoxic in nature. Separation parameters such as the type and concentration of alcohol, acid modifier, and buffer in mobile phase as well as the choice of stationary phase and column temperature were studied. The data indicated that both electrostatic and hydrophilic interactions contributed to the retention and separation of the hydrazines. The results presented here provide insight into the adjustment of the retention and separation of analytes in HILIC mode with alcohol as a weak eluent. The optimized HILIC method coupled with chemiluminescent nitrogen detection (CLND) is simple and sensitive (reporting limit at 0.02%) and was applied to simultaneous analysis of hydrazine and 1,1-dimethylhydrazine in a pharmaceutical intermediate.

  13. Chromatography using a water stationary phase and a carbon dioxide mobile phase.

    PubMed

    Fogwill, Michael O; Thurbide, Kevin B

    2010-12-15

    A novel chromatographic separation method is introduced which employs water (saturated with CO(2)) as a stationary phase and CO(2) (saturated with water) as a mobile phase. Since water and CO(2) have little miscibility, conditions can be attained that create a stationary phase of water lining the inside of an uncoated stainless steel capillary. Because altering temperature and pressure can change both the density of the mobile phase and the polarity of the stationary phase, these experimental parameters offer good flexibility for optimizing separations and allow for different gradient programmed separation options. Further, since this method is free of organic stationary and mobile phase components, it is environmentally compatible and allows the use of universal flame ionization detection. This system offers very good sample capacity, peak symmetry, and retention time reproducibility (∼1% RSD run-to-run, ∼4% RSD day-to-day). Analytes such as alcohols, carboxylic acids, phenols, and tocopherols are employed to investigate this relatively inexpensive and robust method. As an application, the system is used to quantify ethanol in alcoholic beverages and biofuel and to analyze caffeine levels in drinks. In all cases, quantitative results are obtained with quick throughput times and often little need for sample preparation.

  14. Imprinted polymers for chiral resolution of (±)-ephedrine, 4: packed column supercritical fluid chromatography using molecularly imprinted chiral stationary phases.

    PubMed

    Ansell, Richard J; Kuah, Janice K L; Wang, Dongyao; Jackson, Clare E; Bartle, Keith D; Clifford, Anthony A

    2012-11-16

    (-)-Ephedrine-molecularly imprinted polymers (MIPs) have been successfully used as stationary phases in supercritical fluid chromatography for the separation of (±)-ephedrine enantiomers. This approach combines the simple preparation and predictable elution order of MIP stationary phases with the superior mobile phase diffusivity and low viscosity of supercritical fluid mobile phases. The optimised mobile phase comprised supercritical carbon dioxide with a modifier consisting of MeOH/isopropylamine/H(2)O 93:5:2 (v/v/v). In many cases, better resolution separations were observed compared to when liquid mobile phases were used, and better separations achieved at high sample loads, although interestingly the MIPs which work best in SFC are different from the MIPs that work best in HPLC with an amine modifier. The MIP stationary phases were stable under the conditions employed and the chromatography was reproducible. This work opens the door to exploiting MIP stationary phases in preparative SFC.

  15. Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column.

    PubMed

    Padivitage, Nilusha L T; Dissanayake, Milan K; Armstrong, Daniel W

    2013-11-01

    A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van't Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.

  16. Ultra high performance liquid chromatography versus high performance liquid chromatography: stationary phase selectivity for generic carotenoid screening.

    PubMed

    Bijttebier, Sebastiaan; D'Hondt, Els; Noten, Bart; Hermans, Nina; Apers, Sandra; Voorspoels, Stefan

    2014-03-07

    Aim of study was to find the most suitable LC column for generic carotenoid screening. To represent the diversity of carotenoids in nature and to optimize chromatographic separation, a set of carotenoid standards was carefully chosen to account for the various classes of carotenoids. The HPLC C30 column has since long been the 'golden standard' in the chromatographic separation of carotenoids. Since approximately one decade, new UHPLC technology has led to much shorter analysis times, smaller peak widths and higher chromatographic resolution. However, there are currently no UHPLC columns on the market containing the specific stationary phase chemistry of the HPLC C30 column. Therefore during this study, we investigated the separation of carotenoids on a set of UHPLC columns and compared it to their separation on the HPLC C30 column. Comparison of carotenoids separations on the different stationary phases with objective column comparison parameters clearly indicated that the HPLC C30 column is an overall better performer in the separation of carotenoids. This is due to the lack of UHPLC column chemistries that are adapted for carotenoid analysis. However, analysis time on the HPLC C30 column takes about four times longer compared to UHPLC analysis. Therefore, with the range of columns that are commercially available nowadays, a choice has to be made between very high selectivity (HPLC C30 column) and analysis times that are adapted to modern laboratory requirements (UHPLC technology). Therefore, carotenoid separations would be even more performing if an appropriate UHPLC C30 column would be available.

  17. Thermodynamics of the sorption of organic compounds on polyethylene glycol 400-permethylated β-cyclodextrin stationary phase and its enantioselectivity in gas chromatography

    NASA Astrophysics Data System (ADS)

    Kuraeva, Yu. G.; Onuchak, L. A.; Evdokimova, M. A.

    2016-08-01

    The thermodynamic characteristics of sorption of 24 organic compounds of various classes from the gas phase on the binary stationary phase based on polyethylene glycol 400 and permethylated β-cyclodextrin were determined. The influence of geometrical structure and optical activity of organic compounds on the possibility of forming sorbate-macrocycle complexes was examined. It was found that the studied stationary phase shows the enantioselectivity towards low-polar terpenes under the conditions of gas chromatography.

  18. Capillary liquid chromatography and capillary electrochromatography using silica hydride stationary phases.

    PubMed

    Pesek, Joseph J; Matyska, Maria T; Sukul, Dipti

    2008-05-16

    A hydride-based octadecyl stationary phase on both 4.0 and 1.8 microm silica particles is tested in both the capillary LC and the pressurized capillary electrochromatography (pCEC) modes. These two materials are compared to standard C18 stationary phase made by organosilanization and to the hydride material packed into a convention 4.6mm I.D. column. The performance of the capillary columns is evaluated in terms of analysis times for various mixtures as well as efficiency. Of particular interest are the differences between the LC mode where only laminar flow is present and pCEC operation where a flat electrodriven flow profile is superimposed on the parabolic pressurized flow. Differences in performance between columns packed with 4.0 and 1.8 microm particle silica are also evaluated.

  19. Polystyrene-divinylbenzene-glycidyl methacrylate stationary phase grafted with poly (amidoamine) dendrimers for ion chromatography.

    PubMed

    Guo, Dandan; Lou, Chaoyan; Zhang, Peimin; Zhang, Jiajie; Wang, Nani; Wu, Shuchao; Zhu, Yan

    2016-07-22

    In this work, a novel ion exchange stationary phase based on different generations of poly (amidoamine) dendrimers (PAMAM) was developed for the determination of inorganic anions and carbohydrates. Synthesis of the PAMAM was carried out with the polymerization reaction of ethylenediamine and methyl acrylate. The synthesized PAMAM was then grafted to the polystyrene-divinylbenzene-glycidyl methacrylate (PS-GMA) to form PAMAM-based beads. These beads were finally modified with 1,4-butanediol diglycidyl ether (BDDE) to generate the anion exchanger, which were characterized by scanning electron microscopy (SEM), brunauer-emmett-teller (BET), fourier transform infrared spectroscopy (FTIR), and elemental analysis. Elemental analysis, breakthrough curves and capacity factors showed that more epoxy groups and higher PAMAM generations in stationary phase could result in higher anion exchange capacity. The efficiency, durability and stability of the proposed anion exchanger were investigated by using six inorganic anions (fluoride, chloride, nitrite, bromide, nitrate and sulfate) and four carbohydrates (trehalose, glucose, maltotriose and galacturonic acid) as analytes, respectively. The reliability of the proposed ion chromatographic stationary phase was demonstrated by determining the content of galacturonic acid in polysaccharides from Poria cocos and Atractylodes macrocephala. The relative standard deviations of retention time, peak height, and peak area for galacturonic acid were 0.39%, 1.22%, and 2.02%, respectively. The spiked recoveries were in the range of 88.29%-100.51% for plant polysaccharides. Due to the good structural homogeneity, intense internal porosity, biological compatibility and high density of active groups in PAMAM, this grafted stationary phase showed good ion-exchange characteristics, especially in biological charged molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Performance of permethyl pillar[5]arene stationary phase for high-resolution gas chromatography.

    PubMed

    Zhang, Yan; Lv, Qing; Qi, Meiling; Cai, Zhiqiang

    2017-05-05

    This work presents the investigation of permethyl pillar[5]arene (MP5) as stationary phase for capillary gas chromatographic (GC) separations. The MP5 capillary column fabricated by the sol-gel coating method exhibited weak polarity and high column efficiency over 4200 plates/m for n-dodecane, n-octanol and naphthalene. Particularly, the MP5 stationary phase displays unique retention for dibromoalkanes, which was found to be closely related with the linker length, and shows high resolving capability for a wide range of positional and structural isomers, including alkylbenzenes, chlorobenzenes and chloronitrobenzenes, naphthalene derivatives, phenols and anilines. Moreover, the MP5 column showed good thermal stability and repeatability and reproducibility with the relative standard deviation in the range of 0.02-0.04% for intra-day, 0.32-0.46% for inter-day and 1.5-3.4% for between-column, respectively. This work demonstrates an promising future of pillar[n]arenes as a new type of stationary phase in chromatographic separations. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Monitoring the interactions of tocopherol homologues with reversed-phase stationary HPLC phases by 1H suspended-state saturation transfer difference high-resolution/magic angle spinning NMR spectroscopy.

    PubMed

    Schauff, Siri; Friebolin, Volker; Grynbaum, Marc David; Meyer, Christoph; Albert, Klaus

    2007-11-01

    The separation process in reversed-phase high-performance liquid chromatography employing C18 phases is mainly due to hydrophobic interactions. The separation of tocopherol isomers, exhibited by the C30 phases, however, is additionally driven by shape selectivity. This phenomenon is investigated by suspended-state nuclear magnetic resonance spectroscopy using the saturation transfer difference technique, which was originally introduced to study protein-ligand interactions. The interaction strength between beta-/gamma-tocopherol and three different stationary phases was estimated qualitatively. The nuclear magnetic resonance data are compared to chromatographic data, and a similar mode of interaction between the analytes and the stationary phases is elucidated.

  2. Enhanced resolution of Mentha piperita volatile fraction using a novel medium-polarity ionic liquid gas chromatography stationary phase.

    PubMed

    Ragonese, Carla; Sciarrone, Danilo; Grasso, Elisa; Dugo, Paola; Mondello, Luigi

    2016-02-01

    The evaluation of a novel medium-polarity ionic-liquid-based gas chromatography column, SLB-IL60, towards the analysis of a complex essential oil, namely, a peppermint essential oil sample, is reported. The SLB-IL60 30 m column was subjected to bleeding measurements, by means of conventional gas chromatography with mass spectrometry. The SLB-IL60 column was then evaluated in the analysis of pure standard compounds, chosen as typical constituents of peppermint essential oil. Resolution and peak symmetry (expressed as tailing factors at 10% of peak height) were measured and the results were compared to those obtained on the most widely used columns in such an application, namely a medium-polarity [100% poly(ethyleneglycol)] stationary phase, and an apolar 5% diphenyl/95% dimethyl siloxane. The final part of the evaluation was dedicated to the gas chromatography with mass spectrometry analysis of a peppermint essential oil sample and again the data were compared to those obtained on the 100% poly(ethyleneglycol) and the 5% diphenyl/95% dimethyl siloxane phase. Linear retention indices were determined for all the identified components on the ionic liquid capillary. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Preparation and characterization of a new microwave immobilized poly(2-phenylpropyl)methylsiloxane stationary phase for reversed phase high-performance liquid chromatography.

    PubMed

    Begnini, Fernanda R; Jardim, Isabel C S F

    2013-07-05

    A new reversed phase high-performance liquid chromatography (RP-HPLC) stationary phase was prepared and its chromatographic and physical-chemical properties were evaluated. The new stationary phase was prepared with a silica support and poly(2-phenylpropyl)methylsiloxane (PPPMS), a phenyl type polysiloxane copolymer. Since this is a new copolymer and there is little information in the literature, it was submitted to physical-chemical characterization by infrared spectroscopy and thermogravimetry. The chromatographic phase was prepared through sorption and microwave immobilization of the copolymer onto a silica support. The chromatographic performance was evaluated by employing test procedures suggested by Engelhardt and Jungheim, Tanaka and co-workers, Neue, and Szabó and Csató. These test mixtures provide information about the hydrophobic selectivity, silanophilic activity, ion-exchange capacity, shape selectivity and interaction with polar analytes of the new Si-PPPMS reversed phase. Stability tests were developed using accelerated aging tests under both basic and acidic conditions to provide information about the lifetime of the packed columns. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Recent applications of hydrophilic interaction liquid chromatography in pharmaceutical analysis.

    PubMed

    Zhang, Qian; Yang, Feng-Qing; Ge, Liya; Hu, Yuan-Jia; Xia, Zhi-Ning

    2017-01-01

    Hydrophilic interaction liquid chromatography, an alternative liquid chromatography mode, is of particular interest in separating hydrophilic and polar ionic compounds. Compared with traditional liquid chromatography techniques, hydrophilic interaction liquid chromatography offers specific advantages mainly including: (1) relatively green and water-soluble mobile phase composition, which enhances the solubility of hydrophilic and polar ionic compounds; (2) no need for ion-pairing reagents and high content of organic solvent, which benefits mass spectrometry detection; (3) high orthogonality to reverse-phase liquid chromatography, well adapted to two-dimensional liquid chromatography for complicated samples. Therefore, hydrophilic interaction liquid chromatography has been rapidly developed in many areas over the past decades. This review summarizes the recent progress (from 2012 to July 2016) of hydrophilic interaction liquid chromatography in pharmaceutical analysis, with the focus on detecting chemical drugs in various matrices, charactering active compounds of natural products and assessing biotherapeutics through typical structure unit. Moreover, the retention mechanism and behavior of analytes in hydrophilic interaction liquid chromatography as well as some novel hydrophilic interaction liquid chromatography columns used for pharmaceutical analysis are also described.

  5. Retention Characteristics of a pH Tunable Water Stationary Phase in Supercritical Fluid Chromatography.

    PubMed

    Scott, Andrea F; Thurbide, Kevin B

    2017-01-01

    The retention characteristics of a novel pH tunable water stationary phase are presented. The method utilizes a change in mobile phase from N2 to CO2 to acidify the water phase in situ and control the ionization and elution of organic acids. With N2 present the phase pH > 5.4 and the acids are ionized and strongly retained. Conversely, with CO2 present the pH < 3.8 and the acids are neutralized and can elute. This effect is reasonably independent of time. For example, at 80°C hexanoic acid readily elutes from a 10 m column after switching to CO2 at any point over a 1 h period. Beyond this, however, some broadening and peak erosion is noted. Acids are also retained on 10 and 2 m columns similarly, since their elution primarily depends upon the change in stationary phase pH. Altering the CO2 solubility in the water phase alone (i.e., through changing system temperature and pressure without using N2) also produces similar changes in stationary phase acidity. However, this approach yields greater system noise and instability. The N2/CO2 switching mode is used to analyze organic acids in various samples and is found to provide high selectivity for them over other matrix components. Therefore, this approach can potentially simplify the analysis of such acids in complex samples. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Automated sample preparation techniques for the determination of drug enantiomers in biological fluids using liquid chromatography with chiral stationary phases.

    PubMed

    Ceccato, A; Toussaint, B; Chiap, P; Hubert, P; Crommen, J

    1999-01-01

    The determination of drug enantiomers has become of prime importance in the field of pharmaceutical and biomedical analysis. Liquid chromatography (LC) is one of the most frequently used techniques for achieving the separation and quantitation of the enantiomers of drug compounds. In the bioanalytical field, the integrated systems present an interesting alternative to time-consuming sample preparation techniques such as liquid-liquid extraction. Solid phase extraction (SPE) on disposable cartridges, dialysis or column switching are sample preparation techniques that can be fully automated and applied to enantioselective analysis in biological fluids. The selection of the most appropriate LC mode and chiral stationary phase for enantioseparations in bioanalysis is discussed and some aspects of these automated sample preparation procedures are compared, such as selectivity, detectability, elution of the analytes from the extraction sorbent, sample volume and analyte stability.

  7. Nanoengineered analytical immobilized metal affinity chromatography stationary phase by atom transfer radical polymerization: Separation of synthetic prion peptides

    PubMed Central

    McCarthy, P.; Chattopadhyay, M.; Millhauser, G.L.; Tsarevsky, N.V.; Bombalski, L.; Matyjaszewski, K.; Shimmin, D.; Avdalovic, N.; Pohl, C.

    2010-01-01

    Atom transfer radical polymerization (ATRP) was employed to create isolated, metal-containing nanoparticles on the surface of non-porous polymeric beads with the goal of developing a new immobilized metal affnity chromatography (IMAC) stationary phase for separating prion peptides and proteins. Transmission electron microscopy was used to visualize nanoparticles on the substrate surface. Individual ferritin molecules were also visualized as ferritin–nanoparticle complexes. The column's resolving power was tested by synthesizing peptide analogs to the copper binding region of prion protein and injecting mixtures of these analogs onto the column. As expected, the column was capable of separating prion-related peptides differing in number of octapeptide repeat units (PHGGGWGQ), (PHGGGWGQ)2, and (PHGGGWGQ)4. Unexpectedly, the column could also resolve peptides containing the same number of repeats but differing only in the presence of a hydrophilic tail, Q → A substitution, or amide nitrogen methylation. PMID:17481564

  8. Chromatographic evaluation of self-immobilized stationary phases for reversed-phase liquid chromatography.

    PubMed

    Bottoli, Carla B G; Collins, Kenneth E; Collins, Carol H

    2003-02-14

    The preparation of stationary phases for HPLC using polymers deposited on silica usually includes an immobilization step involving cross-linking by free radicals induced by ionizing radiation or by other radical initiators. The present paper reports changes which occur at ambient temperature in the character of poly(methyloctylsiloxane) deposited on porous silica particles as a function of the time interval between particle loading and column packing. Column performance and retention factors increase with time and these changes are attributed to rearrangement (self-assembly) which result in "self-immobilization" of the polymer molecules on the silica surface.

  9. Evaluation of innovative stationary phase ligand chemistries and analytical conditions for the analysis of basic drugs by supercritical fluid chromatography.

    PubMed

    Desfontaine, Vincent; Veuthey, Jean-Luc; Guillarme, Davy

    2016-03-18

    Similar to reversed phase liquid chromatography, basic compounds can be highly challenging to analyze by supercritical fluid chromatography (SFC), as they tend to exhibit poor peak shape, especially those with high pKa values. In this study, three new stationary phase ligand chemistries available in sub -2 μm particle sizes, namely 2-picolylamine (2-PIC), 1-aminoanthracene (1-AA) and diethylamine (DEA), were tested in SFC conditions for the analysis of basic drugs. Due to the basic properties of these ligands, it is expected that the repulsive forces may improve peak shape of basic substances, similarly to the widely used 2-ethypyridine (2-EP) phase. However, among the 38 tested basic drugs, less of 10% displayed Gaussian peaks (asymmetry between 0.8 and 1.4) using pure CO2/methanol on these phases. The addition of 10mM ammonium formate as mobile phase additive, drastically improved peak shapes and increased this proportion to 67% on 2-PIC. Introducing the additive in the injection solvent rather than in the organic modifier, gave acceptable results for 2-PIC only, with 31% of Gaussian peaks with an average asymmetry of 1.89 for the 38 selected basic drugs. These columns were also compared to hybrid silica (BEH), DIOL and 2-EP stationary phases, commonly employed in SFC. These phases commonly exhibit alternative retention and selectivity. In the end, the two most interesting ligands used as complementary columns were 2-PIC and BEH, as they provided suitable peak shapes for the basic drugs and almost orthogonal selectivities.

  10. The pentafluorophenyl stationary phase shows a unique separation efficiency for performing fast chromatography determination of highbush blueberry anthocyanins.

    PubMed

    Šmídová, Barbora; Šatínský, Dalibor; Dostálová, Kateřina; Solich, Petr

    2017-05-01

    A high-performance liquid chromatography method using an alternative pentafluorophenyl (PFP) core-shell stationary phase has been developed and used for rapid separation of 23 anthocyanins in a highbush blueberry Bluehaven cultivar. A high efficiency of separation of anthocyanins was achieved in the core-shell column Kinetex PFP, 150×4.6mm (particle size 2.6µm) with a 5×4.6mm precolumn, using a simple linear gradient elution with a mobile phase of acetonitrile and a water solution of 2% formic acid at a flow rate of 1.0ml/min and at a temperature of 50°C. The detection wavelength was set at 520nm for detection of all anthocyanins. The homogenized blueberry sample (Bluehaven cultivar) was extracted using pure methanol with 1.3% formic acid using an ultrasound bath for 20min and then filtrated. A 5-µL sample volume was directly injected into the HPLC system. The developed method showed an efficient separation of 23 anthocyanins in a total runtime of 21min. The potential of the pentafluorophenyl phase for efficient separation was demonstrated on a wide range of anthocyanins varying in glycosylation and acylation patterns found in highbush blueberries. The fluorinated stationary phase showed an alternative and complementary separation approach providing unique aromatic and polar selectivity in comparison with common C-18 phases. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Preparation of stationary phases for reversed-phase high-performance liquid chromatography using thermal treatments at high temperature.

    PubMed

    Vigna, Camila R M; Bottoli, Carla B G; Collins, Kenneth E; Collins, Carol H

    2007-07-13

    Batches of poly(methyloctylsiloxane) (PMOS)-loaded silica were prepared by deposition from a solution of PMOS into the pores of HPLC silica. Portions of PMOS-loaded silica were subjected to a thermal treatment at 100 degrees C for 24h (condition 1) in a tube furnace under a nitrogen atmosphere. After that, the material was heated for 4h at higher temperatures (150-400 degrees C) (condition 2). Heating at higher temperatures produces polymer bilayers. Non-immobilized and thermally treated stationary phases were characterized by percent carbon, (29)Si cross-polarization magic angle spinning nuclear magnetic resonance spectroscopy and reversed-phase chromatographic performance. The results show that thermal treatment between 150 and 300 degrees C accelerates the immobilization process, possibly due to some bond breaking of the polysiloxane, with formation of strong linkages to the surface of the support, resulting in more complete coverage of the silica. The chromatographic results show an improvement of efficiency with the increase of the temperature of condition 2 up to 300 degrees C and an increase in the resolution of the components, mainly for the phase heated at 300 degrees C. Such results demonstrate that a two-step thermal treatment (100 degrees C then 150-300 degrees C) produces stationary phases with good properties for use in reversed-phase high-performance liquid chromatography.

  12. Polystyrene-divinylbenzene stationary phases agglomerated with quaternized multi-walled carbon nanotubes for anion exchange chromatography.

    PubMed

    Huang, Zhongping; Wu, Hongwei; Wang, Fengli; Yan, Wenwu; Guo, Weiqiang; Zhu, Yan

    2013-06-14

    This work explores the potential of multi-walled carbon nanotubes as an agglomerated material for ion chromatography stationary phases for the separation of inorganic anions. Polyelectrolytes with quaternary ammonium groups were introduced onto the carbon nanotube surface, based on condensation polymerization of 1,4-butanediol diglycidyl ether (BDDE) and methylamine (MA). Quaternized multi-walled carbon nanotubes (Q-MWCNTs) were electrostatically adsorbed onto the surface of sulfonated polystyrene-divinylbenzene (PS-DVB) beads to generate the anion exchanger, which were confirmed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). A 100mm×4.0mm i.d. column was packed with Q-MWCNTs agglomerated PS-DVB particles, with a capacity of 56μequiv./column. Separation of inorganic anions, such as F(-), Cl(-), NO2(-), Br(-), NO3(-), SO4(2-) and PO4(3-) were performed. The stationary phase was rigid, chemically stable and showed good ion-exchange characteristics. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Preparation of hydrophilic interaction/ion-exchange mixed-mode chromatographic stationary phase with adjustable selectivity by controlling different ratios of the co-monomers.

    PubMed

    Bo, Chunmiao; Wang, Xiaomeng; Wang, Chaozhan; Wei, Yinmao

    2017-03-03

    Development of mixed-mode chromatography (MMC) stationary phase with adjustable selectivity is beneficial to meet the needs of complex samples. In this work, surface-initiated atom transfer radical polymerization (SI-ATRP) using the mixture of two functional monomers was proposed as a new preparation strategy for MMC stationary phase with adjustable selectivity. The mixture of sodium 4-styrenesulfonate (NASS) and dimethylaminoethyl methacrylate (DMAEMA) underwent SI-ATRP to bond poly(NASS-co-DMAEMA) on the surface of silica to prepare hydrophilic interaction/ion-exchange mixed-mode stationary phase. Various analytes (neutral, acidic, basic analytes and strong polar nucleosides) were employed to investigate the retention behaviors. The influences of water content and pH of the mobile phase on the retention validated the mixed-mode retention mechanisms of HILIC and ion-exchange. The charge and polarity of stationary phase as well as the separation selectivity were conveniently manipulated by the ratio of NASS to DMAEMA monomer, and the use of DMAEMA in the mixture additionally endowed the column with the temperature-responsive characteristics. Moreover, the application of the developed column was demonstrated by the successful separation of nucleosides, β-agonists and safflower injection. In a word, the proposed strategy can be potentially applied in the controllable preparation of MMC stationary phase with adjustable selectivity.

  14. Orthogonal separation on one beta-cyclodextrin column by switching reversed-phase liquid chromatography and hydrophilic interaction chromatography.

    PubMed

    Feng, Jia-tao; Guo, Zhi-mou; Shi, Hui; Gu, Jiang-ping; Jin, Yu; Liang, Xin-miao

    2010-06-15

    A dual retention combined with reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) has been observed on beta-cyclodextrin (beta-CD) bonded stationary phase. A typical U-shaped retention curve was achieved owing to dual retention mechanism. Based on this observation, a beta-CD column can be operated under reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) modes. Two-dimensional liquid chromatography (2D-LC) analysis can be realized on just a beta-CD column by switching these two different separation modes. In this study, off-line 2D-LC analysis for a natural product was carried out to prove the orthogonal separation between RP-LC and HILIC modes on a Click beta-CD column. Herba Hedyotis Diffusae, the whole grass of Hedyotis Diffusae wild was extracted with water, pretreated with macroporous resin and then first separated at RP-LC mode on the Click beta-CD column to obtain successive fractions, which were then reanalyzed at HILIC mode on the same Click beta-CD column. The result proved that both separation modes on the Click beta-CD column have good retention and peak shape, and these two separation modes have good orthogonality. 2D-LC analysis revealed abundant information in the natural product. Especially numerous minor components were enriched and separated. The mobile phase used in RP-LC and HILIC modes can be same and the switch between these two separation modes is easily realized by changing the ratio of the acetonitrile and water. Hence the mobile phase in this 2D-LC system is completely compatible. This advantage makes this combination is an appropriate 2D-LC method for the solutes having retention at both separation modes.

  15. Size exclusion chromatography of synthetic polymers and biopolymers on common reversed phase and hydrophilic interaction chromatography columns.

    PubMed

    Caltabiano, Anna M; Foley, Joe P; Barth, Howard G

    2016-03-11

    This work describes the applicability of common reversed phase and HILIC columns for size exclusion chromatography of synthetic and natural polymers. Depending on the nature of the solute and column stationary phase, a "non-retention" condition must be created with the aid of the mobile phase to achieve a unique size-based separation in isocratic mode. The various bonded phases show remarkable differences in size separations that are controlled by mobile phase conditions. Polymer-mobile phase and column-mobile phase solvation interactions determine polymer hydrodynamic volume (or solute bulkiness) and polymer-column steric interaction. Solvation interactions in turn depend on polymer, mobile phase and stationary phase polarities. Column-mobile phase solvation interactions determine the structural order of the bonded ligands that can vary from ordered (extended, aligned away from the silica substrate) to disordered (folded, pointing toward the silica substrate). Chain order increases with increased solvent penetration into the bonded phase. Increased chain order reduces pore volume, and therefore decreases the size-separation efficiency of a column. Conversely, decreased chain order increases pore volume and therefore increases the size-separation efficiency. The thermodynamic quality of the mobile phase also plays a significant role in the separation of polymers. "Poor" solvents can significantly reduce the hydrodynamic diameter of a solute and thus change their retention behavior. Medium polarity stationary phases, such as fluoro-phenyl and cyano, exhibit a unique retention behavior. With an appropriate polarity mobile phase, polar and non-polar synthetic polymers of the same molecular masses can be eluted at the same retention volumes.

  16. High-performance liquid chromatography enantioseparation of polyhalogenated 4,4'-bipyridines on polysaccharide-based chiral stationary phases under multimodal elution.

    PubMed

    Peluso, Paola; Mamane, Victor; Aubert, Emmanuel; Cossu, Sergio

    2014-09-01

    An investigation on the high-performance liquid chromatography enantioseparation of 12 polyhalogenated 4,4'-bipyridines on polysaccharide-based chiral stationary phases is described. The overall study was directed toward the generation of efficient separations in order to obtain pure atropisomers that will serve as ligands for building homochiral metal organic frameworks. Four coated columns--namely, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-4, and Lux Amylose-2--and two immobilized columns--namely, Chiralpak IC and IA--were used under normal, polar organic, and reversed-phase elution modes. Moreover, Chiralcel OJ was considered under normal-phase and polar organic conditions. The effect of the chiral selector and mobile phase composition on the enantioseparation, the enantiomer elution order and the beneficial effect of nonstandard solvents were studied. The effect of water in the mobile phase on the enantioselectivity and retention was investigated and retention profiles typical of hydrophilic interaction liquid chromatography were observed. Interesting phenomena of solvent-induced enantiomer elution order reversal occurred under normal-phase mode. All the considered 4,4'-bipyridines were enantioseparated at the multimilligram level.

  17. Thermodynamics of the adsorption of volatile organic compounds in a binary polydimethylsiloxane-permethylated β-cyclodextrin stationary phase, as measured by gas chromatography

    NASA Astrophysics Data System (ADS)

    Onuchak, L. A.; Platonov, V. I.; Kuraeva, Yu. G.

    2013-05-01

    The sorption behavior of 29 organic compounds in a binary polydimethylsiloxane-permethylated β-cyclodextrin stationary phase is investigated using gas chromatography. The effect of the sorbates' polarity, shape, and chirality on the formation of sorbate-cyclodextrin inclusion complexes is studied.

  18. Poly(alkylmethylsiloxanes) thermally immobilized on silica as stationary phases for high-performance liquid chromatography.

    PubMed

    Bottoli, Carla B G; Chaudhry, Zahra F; Fonseca, Dania A; Collins, Kenneth E; Collins, Carol H

    2002-03-01

    Poly(methyloctylsiloxane) (PMOS) and poly(methyloctadecylsiloxane) (PMODS) were sorbed onto porous HPLC silica and thermally immobilized, in the absence of radical initiators, at temperatures in the range of 80 to 180 degrees C. Following extraction of non-immobilized polymer the materials were packed into columns and their chromatographic properties evaluated. The shorter chain (PMOS) stationary phase showed good HPLC characteristics after thermal immobilizations up to 120 degrees C while the longer chain (PMODS) phase gave satisfactory HPLC phases following thermal immobilizations at 80 and 100 degrees C. Stability evaluation for the PMOS and PMODS columns immobilized at 100 degrees C required 250 ml of pH 8.5 mobile phase at 60 degrees C to significantly decrease efficiency, suggesting a long useful life time at neutral pH and ambient temperature.

  19. Separation of stereoisomers of several furan derivatives by capillary gas chromatography-mass spectrometry, supercritical fluid chromatography, and liquid chromatography using chiral stationary phases.

    PubMed

    Kasai, Hiroko F; Tsubuki, Masayoshi; Takahashi, Kazunori; Shirao, Mika; Matsumoto, Yohichiro; Honda, Toshio; Seyama, Yoshiyuki

    2002-11-15

    The direct separation of several stereoisomers (enantiomers and geometrical isomers) of furan derivatives, important intermediates for the synthesis of physiologically active natural products, was achieved using capillary gas chromatography/mass spectrometry with a per-O-methyl-beta-cyclodextrin, supercritical fluid chromatography and high-performance liquid chromatography with a tris(3,5-dimethylphenylcarbamate) of cellulose or amylose for the chiral stational phases, respectively. The temperature dependence of the peak resolution (Rs) and the retention factor (k) over the range of 110-130 degrees was studied using crotyl furfuryl ether in gas chromatography. Successive increases in the Rs value and of the difference between the k value of the E-isomer and the k value of the Z-isomer were observed when the gradient temperature was decreased. The per-O-methyl-beta-cyclodextrin column was suitable for use with volatile furan ethers whose molecular masses are between 150 and 180. In conclusion, the separation of thermally unstable furan derivatives was accomplished using supercritical fluid chromatography and high-performance liquid chromatography.

  20. Microwave-immobilized polybutadiene stationary phase for reversed-phase high-performance liquid chromatography.

    PubMed

    Lopes, Nilva P; Collins, Kenneth E; Jardim, Isabel C S F

    2004-03-19

    Polybutadiene (PBD) has been immobilized on high-performance liquid chromatography (HPLC) silica by microwave radiation at various power levels (52-663 W) and actuation times (3-60 min). Columns prepared from these reversed-phase HPLC materials, as well as from similar non-irradiated materials, were tested with standard sample mixtures and characterized by elemental analysis (%C) and infrared spectroscopy. A microwave irradiation of 20 min at 663 W gives a layer of immobilized PBD that presented good performance. Longer irradiation times give thicker immobilized layers having less favorable chromatographic properties.

  1. Crosslinked structurally-tuned polymeric ionic liquids as stationary phases for the analysis of hydrocarbons in kerosene and diesel fuels by comprehensive two-dimensional gas chromatography.

    PubMed

    Zhang, Cheng; Park, Rodney A; Anderson, Jared L

    2016-04-01

    Structurally-tuned ionic liquids (ILs) have been previously applied as the second dimension column in comprehensive two-dimensional gas chromatography (GC×GC) and have demonstrated high selectivity in the separation of individual aliphatic hydrocarbons from other aliphatic hydrocarbons. However, the maximum operating temperatures of these stationary phases limit the separation of analytes with high boiling points. In order to address this issue, a series of polymeric ionic liquid (PIL)-based stationary phases were prepared in this study using imidazolium-based IL monomers via in-column free radical polymerization. The IL monomers were functionalized with long alkyl chain substituents to provide the needed selectivity for the separation of aliphatic hydrocarbons. Columns were prepared with different film thicknesses to identify the best performing stationary phase for the separation of kerosene. The bis[(trifluoromethyl)sulfonyl]imide ([NTf2](-))-based PIL stationary phase with larger film thickness (0.28μm) exhibited higher selectivity for aliphatic hydrocarbons and showed a maximum allowable operating temperature of 300°C. PIL-based stationary phases containing varied amount of IL-based crosslinker were prepared to study the effect of the crosslinker on the selectivity and thermal stability of the resulting stationary phase. The optimal resolution of aliphatic hydrocarbons was achieved when 50% (w/w) of crosslinker was incorporated into the PIL-based stationary phase. The resulting stationary phase exhibited good selectivity for different groups of aliphatic hydrocarbons even after being conditioned at 325°C. Finally, the crosslinked PIL-based stationary phase was compared with SUPELCOWAX 10 and DB-17 columns for the separation of aliphatic hydrocarbons in diesel fuel. Better resolution of aliphatic hydrocarbons was obtained when employing the crosslinked PIL-based stationary phase as the second dimension column. Copyright © 2016 Elsevier B.V. All rights

  2. [Systematic evaluation of retention behavior of carbohydrates in hydrophilic interaction liquid chromatography].

    PubMed

    Fu, Qing; Wang, Jun; Liang, Tu; Xu, Xiaoyong; Jin, Yu

    2013-11-01

    A systematic evaluation of retention behavior of carbohydrates in hydrophilic interaction liquid chromatography (HILIC) was performed. The influences of mobile phase, stationary phase and buffer salt on the retention of carbohydrates were investigated. According to the results, the retention time of carbohydrates decreased as the proportion of acetonitrile in mobile phase decreased. Increased time of carbohydrates was observed as the concentration of buffer salt in mobile phase increased. The retention behavior of carbohydrates was also affected by organic solvent and HILIC stationary phase. Furthermore, an appropriate retention equation was used in HILIC mode. The retention equation lnk = a + blnC(B) + cC(B) could quantitatively describe the retention factors of carbohydrates of plant origin with good accuracy: the relative error of the predicted time to actual time was less than 0.3%. The evaluation results could provide guidance for carbohydrates to optimize the experimental conditions in HILIC method development especially for carbohydrate separation

  3. Carboxylate modified porous graphitic carbon: a new class of hydrophilic interaction liquid chromatography phases.

    PubMed

    Wahab, M Farooq; Ibrahim, Mohammed E A; Lucy, Charles A

    2013-06-18

    Stationary phases for hydrophilic interaction liquid chromatography (HILIC) are predominantly based on silica and polymer supports. We present porous graphitic carbon particles with covalently attached carboxylic acid groups (carboxylate-PGC) as a new HILIC stationary phase. PGC particles were modified by adsorbing the diazonium salt of 4-aminobenzoic acid onto the PGC, followed by reduction of the adsorbed salt with sodium borohydride. The newly developed carboxylate-PGC phase exhibits different selectivity than that of 35 HPLC columns, including bare silica, zwitterionic, amine, reversed, and unmodified PGC phases. Carboxylate-PGC is stable from pH 2.0 to 12.6, yielding reproducible retention even at pH 12.6. Characterization of the new phase is presented by X-ray photoelectron spectroscopy, thermogravimetry, zeta potentials, and elemental analysis. The chromatographic performance of carboxylate-PGC as a HILIC phase is illustrated by separations of carboxylic acids, nucleotides, phenols, and amino acids.

  4. Anion separations for liquid chromatography using propylpyridinium silica as the stationary phase.

    PubMed

    Auler, Lúcia M L A; Silva, César R; Bottoli, Carla B G; Collins, Carol H

    2011-05-30

    This work describes the characterization and potential applications of a silica-based anion-exchange phase prepared by a two-step modification process that incorporates a propylpyridinium group. The effects of pH and eluent concentration on anion separation were examined using 150 mm × 3.9 mm HPLC columns packed with the new phase. The mobile phase pH values ranged from 3.8 to 6.6 using phthalic acid/Tris solutions. The best separation was achieved using 2.5 mmol L(-1) phthalate/2.4 mmol L(-1) Tris solution at pH 4.2 as mobile phase with non-suppressed conductivity detection. The new stationary phase was used for the separation of some inorganic and organic anions showing good resolution. The stability of the silica-based anion exchange phase was also evaluated. Analytical curves, for concentrations ranging from 0.25 to 10 mg L(-1) for the inorganic anions chloride, nitrite, bromide and nitrate, showed good linear correlations (r>0.998). The method was tested with certified rainwater samples. The measured and certified values were in good agreement, indicating that the new phase holds significant promise for the analysis of these anions in environmental samples.

  5. Super/subcritical fluid chromatography chiral separations with macrocyclic glycopeptide stationary phases.

    PubMed

    Liu, Ying; Berthod, Alain; Mitchell, Clifford R; Xiao, Tom Ling; Zhang, Bo; Armstrong, Daniel W

    2002-11-29

    The chiral recognition capabilities of three macrocyclic glycopeptide chiral selectors, namely teicoplanin (Chirobiotic T), its aglycone (Chirobiotic TAG) and ristocetin (Chirobiotic R), were evaluated with supercritical and subcritical fluid mobile phases. A set of 111 chiral compounds including heterocycles, analgesics (nonsteroidal antiinflamatory compounds), beta-blockers, sulfoxides, N-protected amino acids and native amino acids was separated on the three chiral stationary phases (CSPs). All separations were done with an outlet pressure regulated at 100 bar, 31 degrees C and at 4 ml/min. Various amounts of methanol ranging from 7 to 67% (v/v) were added to the carbon dioxide along with small amounts (0.1 to 0.5%, v/v) of triethylamine and/or trifluoroacetic acid. The Chirobiotic TAG CSP was the most effective closely followed by the Chirobiotic T column. Both columns were able to separate, partially or fully, 92% of the enantiomers of the compound set. The ristocetin chiral selector could partially or baseline resolve only 60% of the enantiomers tested. All separations were done in less than 15 min and 70% were done in less than 4 min. The speed of the separations is the main advantage of the use of SFC compared to normal-phase HPLC. In addition, SFC is advantageous for preparative separations with easy solute recovery and solvent disposal.

  6. Aminoglycoside analysis in food of animal origin with a zwitterionic stationary phase and liquid chromatography-tandem mass spectrometry.

    PubMed

    Díez, Cristina; Guillarme, Davy; Staub Spörri, Aline; Cognard, Emmanuelle; Ortelli, Didier; Edder, Patrick; Rudaz, Serge

    2015-07-02

    In this study, fourteen highly polar aminoglycoside (AGs) antibiotics were selected. Various stationary phases were tested, including Obelisc R, ZIC-HILIC, BEH amide and aminopropyl. The nature of the stationary phase, mobile phase (water or buffer solutions and acetonitrile), pH (percentage of formic acid), gradient conditions and injection solvents were systematically studied as relevant parameters for tuning retention selectivity and detectability of AGs in liquid chromatography electrospray tandem mass spectrometry (LC-(ESI)-MS/MS). Only the two zwitterionic phases (Obelisc R and ZIC-HILIC) achieved a proper chromatographic separation considering interferences due to the crosstalk effect in low resolution mass spectrometers. The water/acetonitrile mobile phase containing 1% formic acid used with Obelisc R provided more sensitivity than the highly concentrated buffered mobile phases required for ZIC-HILIC. A solid phase extraction (SPE) clean-up procedure with polymeric weak cation exchange (WCX) cartridges was optimized for honey, milk and liver samples. Different brands of cartridges and elution solvents were tested, and the Taurus WCX offered the best recovery rate with a buffer elution at pH 3. The final optimized method was validated in these matrices according to Decision 2002/657/EC. A monitoring campaign for sixty honey, milk and liver samples was carried out at the Food Authority Control in Geneva. The concentration of dihydrostreptomycin (DSTP) found in one ovine liver exceeded the established maximum residue levels (MRLs) within the European and Swiss legislations but it was compliant taking into account the validation data. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. In situ synthesis of metal-organic frameworks in a porous polymer monolith as the stationary phase for capillary liquid chromatography.

    PubMed

    Yang, Shengchao; Ye, Fanggui; Zhang, Cong; Shen, Shufen; Zhao, Shulin

    2015-04-21

    In this study, HKUST-1 was synthesized in situ on the porous polymer monolith as the stationary phase for capillary liquid chromatography (cLC). The unique carboxyl functionalized poly(methacrylic acid-co-ethylene dimethacrylate) (poly(MAA-co-EDMA)) monolith was used as a support to directly grow HKUST-1 by a controlled layer-by-layer self-assembly strategy. X-ray diffraction, scanning electron microscopy, energy dispersive X-ray spectrometry, and Fourier transform infrared spectroscopy of the resulting HKUST-1-poly(MAA-co-EDMA) monoliths indicated that HKUST-1 was successfully grafted onto the pore surface of the poly(MAA-co-EDMA) monolith. The column performance of HKUST-1-poly(MAA-co-EDMA) monoliths for the separation of various small molecules, such as benzenediols, xylenes, ethylbenzenes, and styrenes, was evaluated. The chromatographic performance was found to improve with increasing HKUST-1 density, and the column efficiencies and resolutions of HKUST-1-poly(MAA-co-EDMA) monoliths were 18 320-19 890 plates m(-1) and 1.62-6.42, respectively, for benzenediols. The HKUST-1-poly(MAA-co-EDMA) monolith displayed enhanced resolution for the separation of positional isomers when compared to the traditional C18 and HKUST-1 incorporated polymer monoliths. Hydrophobic, π-π, and hydrogen bonding interactions within the HKUST-1-poly(MAA-co-EDMA) monolith were observed in the separation of small molecules. The results showed that the HKUST-1-poly(MAA-co-EDMA) monoliths are promising stationary phases for cLC.

  8. Separation techniques: Chromatography

    PubMed Central

    Coskun, Ozlem

    2016-01-01

    Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification. PMID:28058406

  9. Preparation and evaluation of bonded linear polymethacrylate stationary phases for open tubular capillary electrokinetic chromatography

    SciTech Connect

    Tan, Z.J.; Remcho, V.T.

    1997-02-15

    A new procedure for the preparation of thick polymethacrylate films bonded in 25 {mu}m i.d. fused-silica capillaries is developed. The etched silica surface is first modified with an unsaturated organosilane, which is later incorporated into the polymer film. The capillary is then filled with a monomer solution, and polymerization is initiated by incubation at elevated temperature. This thermoinitiation method enables the use of ordinary polyimide-jacketed capillaries in preparing the columns. The effect of monomer concentration on the resulting polymer film was studied by open tubular capillary electrokinetic chromatography using p-hydroxybenzoates (parabens) as test solutes. Good separations were achieved using short capillaries. Run-to-run retention time reproducibility was excellent, with RSDs of 2% (n = 50) being representative. For the linear polymer films produced, retention of analytes increased as the monomer concentration increased to a certain value, at which point the capacity factors level off with further increases in monomer concentration. The electroosmotic flow velocity decreases with increasing monomer concentration. The efficiency for an unretained test probe (acetone) reaches 270 000 plates/m. 13 refs., 10 figs., 1 tab.

  10. Sulfonated acrylamide copolymers as pseudo-stationary phases in electrokinetic chromatography.

    PubMed

    Shi, W; Watson, C J; Palmer, C P

    2001-01-05

    Sulfonated copolymers were synthesized, characterized and used as separation media in electrokinetic chromatography. The polymers used were synthesized from AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) and LMAm (lauryl methacrylamide) in different mole ratios (from 100:0 to 60:40). Electrophoretic mobilities and methylene selectivities were calculated, which showed the expected correlation with the monomer ratios. The chemical selectivities for the separation of nine solutes by the copolymers were compared with that of sodium lauryl sulfate micelles, showing significant differences. No significant difference in chemical selectivities was observed for copolymers with different monomer ratios. No significant change of hydrophobic microdomain of copolymers was found in background buffers with different ionic strength values, based on the investigation of the retention factors, methylene selectivities and polymer effective mobilities. No change of hydrophobic microdomain of the copolymer solutions was found at copolymer concentrations from 0.17 to 3% (w/v), however, plots of k' versus polymer concentration suggested a different copolymer phase at lower concentrations (from 0 to 0.1%, w/v) from that at higher concentrations (from 0.17 to 3%, w/v). The copolymer with AMPS-LMAm (80:20) could be chosen as optimum copolymer as far as the methylene selectivity, peak symmetry and polymer mobility were concerned.

  11. Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

    PubMed

    Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

    2015-08-01

    Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Characterization and classification of pseudo-stationary phases in micellar electrokinetic chromatography using chemometric methods.

    PubMed

    Fu, Cexiong; Khaledi, Morteza G

    2014-03-04

    Two types of chemometric methods, principal component analysis (PCA) and cluster analysis, are employed to characterize and classify a total of 70 pseudostationary phases (54 distinct systems and 16 decoy systems) in micellar electrokinetic chromatography (MEKC). PCA excels at removing redundant information for micellar phase characterization and retaining principal determinants for phase classification. While PCA is useful in the characterization of micelle selectivities, it is ineffective in defining the grouping of micellar phases. Hierarchical clustering yields a complete dendrogram of cluster structures but provides only limited cluster characterizations. The combination of these two chemometric methods leads to a comprehensive interpretation of the micellar phase classification. Moreover, the k-means analysis can further discern subtle differences among those closely located micellar phases. All three chemometric methods result in similar classifications with respect to the similarities and differences of the 70 micelle systems investigated. These systems are categorized into 3 major clusters: fluoro-surfactants represent cluster I, identified as strong hydrogen bond donors and dipolar but weak hydrogen bond acceptors. Cluster II includes sulfonated acrylamide/acrylate copolymers and surfactants with trimethylammonium head groups, characterized by strong hydrophobicity (v) and weak hydrogen bond acidity (b). The last cluster consists of two subclusters: clusters III and IV. Cluster III includes siloxane-based polymeric micelles, exhibiting weak hydrophobicity and medium hydrogen bond acidity and basicity (a), and the cluster IV micellar systems are characterized by their strong hydrophobicity and medium hydrogen bond acidity and basicity but rather weak dipolarity. Cluster III differs from cluster IV by its slightly weaker hydrophobicity and hydrogen bond donating capability. The classification by chemometric methods is in good agreement with the

  13. Titanium-scaffolded organic-monolithic stationary phases for ultra-high-pressure liquid chromatography.

    PubMed

    Vonk, Rudy J; Vaast, Axel; Eeltink, Sebastiaan; Schoenmakers, Peter J

    2014-09-12

    Organic-polymer monoliths with overall dimensions larger than one millimetre are prone to rupture - either within the monolith itself or between the monoliths and the containing wall - due to the inevitable shrinkage accompanying the formation of a cross-linked polymeric network. This problem has been addressed by creating titanium-scaffolded poly(styrene-co-divinylbenzene) (S-co-DVB) monoliths. Titanium-scaffolded monoliths were successfully used in liquid chromatography at very high pressures (up to 80MPa) and using gradients spanning the full range of water-acetonitrile compositions (0 to 100%). The kinetic-performance of (50-mm long) titanium-scaffolded monoliths was compared to that of similar monolith created in 1-mm i.d. glass-lined tubing at pressures up to 50MPa. The peak capacities obtained with the titanium-scaffolded column was about 30% lower. An increased Eddy-diffusion, due to the pillar-structure, and a decreased permeability are thought to be the main reasons for this reduced kinetic-performance. No decrease in performance was observed when the titanium-scaffolded columns were operated at pressures of 80MPa for up to 12h. The column-to-column repeatability (n=5) was acceptable in terms of observed peak widths at half heights (RSD ca. 10%) The run-to-run repeatability (n=135) in terms of retention times and peak widths at half height were found to be good. Titanium-scaffolded columns coupled in series up to a combined length of (200mm) were used for the analyses of a complex Escherichia coli protein sample. Our experiments demonstrate that columns based on titanium-scaffolded organic-polymer monolith can be operated under strenuous conditions without loss in performance. The titanium-scaffolded approach makes it feasible to create organic-polymer monoliths in wide-bore columns with accurate temperature control.

  14. Polymer-coated fibrous materials as the stationary phase in packed capillary gas chromatography.

    PubMed

    Saito, Yoshihiro; Tahara, Ai; Imaizumi, Motohiro; Takeichi, Tsutomu; Wada, Hiroo; Jinno, Kiyokatsu

    2003-10-15

    Synthetic polymer filaments have been introduced as the support material in packed capillary gas chromatography (GC). The filaments of the heat-resistant polymers, Zylon, Kevlar, Nomex, and Technora, were longitudinally packed into a short fused-silica capillary, followed by the conventional coating process for open-tubular GC columns. The separation of several test mixtures such as n-alkylbenzenes and n-alkanes was carried out with these polymer-coated fiber-packed capillary columns. With the coating by various polymeric materials on the surface of these filaments, the retentivity was significantly improved over the parent fiber-packed column (without polymer coating) as well as a conventional open-tubular capillary of the same length. The results demonstrated a good combination of Zylon as the support and poly(dimethylsiloxane)-based materials as the coating liquid-phase for the successful GC separation of n-alkanes and polycyclic aromatic hydrocarbons (PAHs), while successful applications for other separations such as poly(ethylene glycol) coating for the separation of alcohols were also obtained. From the results it has been suggested that the selectivity of the fiber-packed column could be tuned by selecting different coating materials, indicating the promising possibility for a novel usage of fine fibrous polymers as the support material that can be combined with newly synthesized coating materials specially designed for particular separations. Taking advantage of good thermal stability of the fibers, the column temperature could be elevated to higher than 350 degrees C with the combination of a short metallic capillary.

  15. Peptide separation by Hydrophilic-Interaction Chromatography: a review.

    PubMed

    Yoshida, Tatsunari

    2004-09-30

    Recent developments in the separation of peptides by high-performance liquid chromatography (HPLC) using polar sorbents with less polar eluents are summarized in this review. This separation mode is now commonly referred to as Hydrophilic-Interaction Chromatography (HILIC). The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions are studied on TSKgel Amide-80 columns, which consist of carbamoyl groups bonded to a silica gel matrix, using a mixture of acetonitrile (MeCN)-water containing 0.1% trifluoroacetic acid (TFA). Some applications are given in peptide field using Hydrophilic-Interaction Chromatography.

  16. Enantioselective determination of protein amino acids in fertilizers by liquid chromatography-tandem mass spectrometry on chiral teicoplanin stationary phase.

    PubMed

    Taujenis, Lukas; Olšauskaitė, Vilma; Padarauskas, Audrius

    2014-11-19

    High-performance liquid chromatography on a glycopeptide antibiotic teicoplanin-based chiral stationary phase coupled with tandem mass spectrometry was developed for fast and reliable enantioseparation and determination of protein amino acids in hydrolyzed fertilizer samples. The effect of the mobile phase parameters (type and content of organic modifier and pH) and the column temperature on the enantioselectivity was investigated. Under optimized conditions, the majority (15 of 19) of d/l-amino acid pairs were resolved with a resolution factor (Rs) higher than 1.5 with a run time of 15 min. A triple quadrupole tandem mass spectrometer operating in multiple reaction monitoring mode with an electrospray ionization (ESI) ion source was employed for detection. The method was validated in terms of linearity, limits of detection, limits of quantitation, precision, and accuracy. Linear responses were obtained with determination coefficients higher than 0.998 for all analytes, and limits of detection were from 0.04 to 0.24 μg/mL. Sample spike/recovery experiments gave recovery values ranging from 73% for d-threonine to 116% for L-tryptophan. Relative standard deviations for inter- and intraday precision experiments were lower than 21.7%. The developed method was successfully applied for determination of the free amino acid enantiomers in five commercially available hydrolyzed protein fertilizer samples.

  17. Synthesis of novel chiral imidazolium stationary phases and their enantioseparation evaluation by high-performance liquid chromatography.

    PubMed

    Wang, Tao; Yang, Haiyan; Qiu, Ruchen; Huang, Shaohua

    2016-11-09

    Two novel chiral stationary phases (CSPs) were prepared by bonding chiral imidazoliums on the surface of silica gel. The chiral imidazoles were derivatized from chiral amines, 1-phenylethylamine and 1-(1-naphthyl)ethylamine. The obtained CSPs were characterized by Fourier Transform Infrared (FT-IR) spectroscopy and elemental analysis (EA), demonstrating the bonding densities of CSP 1 and CSP 2 were 0.43 mmol g(-1) and 0.40 mmol g(-1), respectively. These two CSPs could be used to availably separate 8 pharmaceuticals, 7 mandelic acid/its derivatives, 2 1-phenylethylamine derivatives, 1 1,1'-bi-2-naphthol, and 1 camphorsulfonic acid in high-performance liquid chromatography (HPLC). It is found that CSP 1 could effectively enantioseparate most chiral analytes, especially the acidic components, while CSP 2 could enantiorecognize all chiral analytes, although a number of components did not achieve baseline separation. Additionally, the effects of mobile phase composition, mobile phase pH and salt content, chiral selector structures, and analyte structures on the enantiorecognitions of the two CSPs were investigated. It is found that high acetonitrile content in mobile phases was conducive to enantiorecognition. Mobile phase pH and salt content could alter the retention behaviors of different enantiomers of the same chiral compound, resulting in better enantioresolution. Moreover, both chiral selector structures and substituted groups of analytes played a significant role in the separation of chiral solutes.

  18. Optimization of milk odd and branched-chain fatty acids analysis by gas chromatography using an extremely polar stationary phase.

    PubMed

    Gómez-Cortés, P; Rodríguez-Pino, V; Juárez, M; de la Fuente, M A

    2017-09-15

    Odd and branched-chain fatty acids (OBCFA) are of interest, since they have bioactive properties and could be regarded biomarkers of ruminant fat intake. An accurate analysis of the individual OBCFA in milk by gas chromatography (GC) is not easy due to milk fat complexity. The availability of ionic liquid stationary phases as SLB-IL111 can be a useful tool to discriminate OBCFA from other milk FA eluting in the same chromatographic regions. The elution behavior of OBCFA on SLB-IL111 was evaluated based on different GC oven temperature programs. All programs assayed discriminated 11:0, iso 13:0, anteiso 13:0, iso 15:0, anteiso 15:0, 15:0 and iso 17:0. Using an initial temperature of 150°C for 1h, 13:0 and iso 16:0 were separated from trans-12:1 and 13-14:1, respectively, whereas iso 18:0 was discriminated from cis-16:1 isomers. 17:0 and 21:0 were well resolved only when an initial GC temperature of 160°C was applied. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Application of polymer based stationary phases in high performance liquid chromatography and capillary high performance liquid chromatography hyphenated to microcoil 1H nuclear magnetic resonance spectroscopy.

    PubMed

    Grynbaum, Marc David; Meyer, Christoph; Putzbach, Karsten; Rehbein, Jens; Albert, Klaus

    2007-07-13

    The increased demand for chromatographic materials that are able to achieve a fast separation of large quantities of structure analogues is a great challenge. It is known that polymer based chromatographic materials have a higher loadability, compared to silica based sorbents. Unfortunately these polymer materials cannot be used under high pressure which is necessary in order to obtain high flow rates, and hence long times are needed to perform a separation. However, by immobilizing a polymer on a mechanically stable porous silica core, this problem can be circumvented and higher flows become feasible on these materials. Especially for capillary liquid chromatography hyphenated with nuclear magnetic resonance a high loadability is of great importance in order to obtain sharp, resolved, and concentrated peaks thus resulting in a good signal to noise ratio in the NMR experiment. Therefore, a highly shape selective chromatographic sorbent was developed by covalently immobilizing a poly(ethylene-co-acrylic) acid copolymer (-CH(2)CH(2)-)(x)[CH(2)CH(CO(2)H)-](y) (x=119, y=2.4) with a mass fraction of acrylic acid of 5% as stationary phase on silica via a spacer molecule (3-glycidoxypropyltrimethoxysilane). First, the loadability of this sorbent compared to C(30) is demonstrated by the HPLC separation of two xanthophyll isomers. Subsequently, it has been successfully employed in the hyphenation of capillary HPLC with microcoil (1)H NMR spectroscopy by separating and identifying a highly concentrated solution of the tocopherol homologues.

  20. Evaluation of different hydrophilic stationary phases for the simultaneous determination of iminosugars and other low molecular weight carbohydrates in vegetable extracts by liquid chromatography tandem mass spectrometry.

    PubMed

    Rodríguez-Sánchez, S; Quintanilla-López, J E; Soria, A C; Sanz, M L

    2014-11-01

    Iminosugars are considered potential drug candidates for the treatment of several diseases, mainly as a result of their α-glycosidase inhibition properties. A method by hydrophilic interaction liquid chromatography tandem mass spectrometry has been optimized for the first time for the simultaneous determination of complex mixtures of bioactive iminosugars and other low molecular weight carbohydrates (LMWC) in vegetable extracts. Three hydrophilic stationary phases (sulfoalkylbetaine zwitterionic, polyhydroxyethyl aspartamide and ethylene bridge hybrid (BEH) with trifunctionally bonded amide) were compared under both basic and acidic conditions. The best sensitivity (limits of detection between 0.025 and 0.28ngmL(-1)) and overall chromatographic performance in terms of resolution, peak width and analysis time were obtained with the BEH amide column using 0.1% ammonium hydroxide as a mobile phase additive. The optimized method was applied to the analysis of extracts of hyacinth bulbs, buckwheat seeds and mulberry leaves. Iminosugar and other LMWC structures were tentatively assigned by their high resolution daughter ions mass spectra. Several iminosugars such as glycosyl-fagomine in mulberry extract were also described for the first time. Among the extracts analysed, mulberry showed the widest diversity of iminosugars, whereas the highest content of them was found in hyacinth bulb (2.5mgg(-1)) followed by mulberry (1.95 mgg(-1)). Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Preparation and application of methylcalix[4]resorcinarene-bonded silica particles as chiral stationary phase in high-performance liquid chromatography.

    PubMed

    Tan, Huey Min; Soh, Shu Fang; Zhao, Jia; Yong, E L; Gong, Yinhan

    2011-01-01

    Two new types of methylcalix[4]resorcinarene-bonded stationary phases, (3-(C-methylcalix[4]resorcinarene)-2-hydroxypropoxy)-propylsilyl-appended silica particles (MCR-HPS) and bromoacetate-substituted MCR-HPS particles (BAMCR-HPS), have been synthesized and used as chiral stationary phases for high-performance liquid chromatography (HPLC) for the first time. The synthetic stationary phases are characterized by means of elemental analysis and Fourier-transform infrared spectroscopy. The chromatographic behavior of MCR-HPS and BAMCR-HPS was studied with several disubstituted benzenes and some chiral drug compounds under both normal phase and reversed-phase conditions. The results show that MCR-HPS has excellent selectivity for the separation of aromatic positional isomers and BAMCR-HPS exhibits excellent performance for separation of enantiomers of chiral compounds.

  2. Separation of opiate alkaloids by electrokinetic chromatography with sulfated-cyclodextrin as a pseudo-stationary phase.

    PubMed

    Zakaria, Philip; Macka, Miroslav; Haddad, Paul R

    2003-01-24

    The separation of six related opiate alkaloids (morphine, thebaine, 10-hydroxythebaine, codeine, oripavine and laudanine) was studied using sulfated-cyclodextrin (s-CD) as a cation-exchange pseudo-stationary phase. Cation-exchange interactions between the cationic analytes and the anionic s-CD (7-11 mol of sulfate groups per mole CD) were found to bethe predominant mechanism, allowing the separations to be performed at low pH where the opiates are protonated and exhibit very similar mobilities. The concentrations of the s-CD and the competing ion (Na+ or Mg2+) in the electrolyte were used to govern the extent of the ion-exchange interactions. Interactions with the sulfated-cyclodextrin differed for each analyte, with oripavine exhibiting the strongest interaction and 10-thebaine and laudanine showing the weakest interactions. Despite the very similar structures of the analytes, these differences resulted in significant changes in separation selectivity. The separation was modelled using a migration equation derived from first principles and based on ion-exchange interactions between the s-CD and the opiates. Constants within the model were obtained by non-linear regression using a small subset of experimentally determined migration times. These constants related to the ion-exchange affinities of the s-CD for the various opiates. When the model was used to predict migration times under other experimental conditions, a very good correlation was obtained between observed and predicted mobilities (r2=0.996). Optimisation of the system was performed using the normalised resolution product and minimum resolution criteria and this process provided two optimised separations, each exhibiting a different separation selectivity.

  3. Strong cation exchange-type chiral stationary phase for enantioseparation of chiral amines in subcritical fluid chromatography.

    PubMed

    Wolrab, Denise; Kohout, Michal; Boras, Mario; Lindner, Wolfgang

    2013-05-10

    A new strong cation exchange type chiral stationary phase (SCX CSP) based on a syringic acid amide derivative of trans-(R, R)-2-aminocyclohexanesulfonic acid was applied to subcritical fluid chromatography (SFC) for separation of various chiral basic drugs and their analogues. Mobile phase systems consisting of aliphatic alcohols as polar modifiers and a broad range of amines with different substitution patterns and lipophilicity were employed to evaluate the impact on the SFC retention and selectivity characteristics. The observed results point to the existence of carbonic and carbamic acid salts formed as a consequence of reactions occurring between carbon dioxide, the alcoholic modifiers and the amine species present in the sub/supercritical fluid medium, respectively. Evidence is provided that these species are essential for affecting ion exchange between the strongly acidic chiral selector units and the basic analytes, following the well-established stoichiometric displacement mechanisms. Specific trends were observed when different types of amines were used as basic additives. While ammonia gave rise to the formation of the most strongly eluting carbonic and carbamic salt species, simple tertiary amines consistently provided superior levels of enantioselectivity. Furthermore, trends in the chiral SFC separation characteristics were investigated by the systematic variation of the modifier content and temperature. Different effects of additives are interpreted in terms of changes in the relative concentration of the transient ionic species contributing to analyte elution, with ammonia-derived carbamic salts being depleted at elevated temperatures by decomposition. Additionally, in an effort to optimize SFC enantiomer separation conditions for selected analytes, the impact of the type of the organic modifier, temperature, flow rate and active back pressure were also investigated.

  4. Cation-exchange chromatography of monoclonal antibodies: characterisation of a novel stationary phase designed for production-scale purification.

    PubMed

    Urmann, Marina; Graalfs, Heiner; Joehnck, Matthias; Jacob, Lothar R; Frech, Christian

    2010-01-01

    A novel cation-exchange resin, Eshmuno™ S, was compared to Fractogel® SO3(-) (M) and Toyopearl GigaCap S-650M. The stationary phases have different base matrices, and carry specific types of polymeric surface modifications. Three monoclonal antibodies (mAbs) were used as model proteins to characterize these chromatographic resins. Results from gradient elutions, stirred batch adsorptions and confocal laser scanning microscopic investigations were used to elucidate binding behaviour of mAbs onto Eshmuno™ S and Fractogel® SO3(-) and the corresponding transport mechanisms on these two resins. The number of charges involved in mAb binding for Eshmuno™ S is lower than for Fractogel® SO3(-), indicating a slightly weaker electrostatic interaction. Kinetics from batch uptake experiments are compared to kinetic data obtained from confocal laser scanning microscopy images. Both experimental approaches show an accelerated protein adsorption for the novel stationary phase. The influence of pH, salt concentrations and residence times on dynamic binding capacities was determined. A higher dynamic binding capacity for Eshmuno™ S over a wider range of pH values and residence times was found compared to Fractogel® SO3(-) and Toyopearl GigaCap S-650M. The capture of antibodies from cell culture supernatant, as well as post-protein A eluates, were analyzed with respect to their host cell protein (hcp) removal capabilities. Comparable or even better hcp clearance was observed at much higher protein loading for Eshmuno™ S than Fractogel® SO3(-) or Toyopearl GigaCap S-650M.

  5. Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for Liquid Chromatography. Part II: Chemical and Thermal Stability.

    PubMed

    Borges, Endler M; Volmer, Dietrich A

    2015-08-01

    In the first part of this review, stationary phases (silica, hybrid silica, hydride silica and non-silica stationary phases) were characterized and compared with respect to selectivity, efficiency, resolution, solvent consumption and analysis time. The present review focuses on the thermal and chemical stability of stationary phases. Stationary phases of high chemical and thermal stability are required for separations that are carried over a wide pH and/or temperature range. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Selectivity differences of water-soluble vitamins separated on hydrophilic interaction stationary phases.

    PubMed

    Yang, Yuanzhong; Boysen, Reinhard I; Hearn, Milton T W

    2013-06-01

    In this study, the retention behavior and selectivity differences of water-soluble vitamins were evaluated with three types of polar stationary phases (i.e. an underivatized silica phase, an amide phase, and an amino phase) operated in the hydrophilic interaction chromatographic mode with ESI mass spectrometric detection. The effects of mobile phase composition, including buffer pH and concentration, on the retention and selectivity of the vitamins were investigated. In all stationary phases, the neutral or weakly charged vitamins exhibited very weak retention under each of the pH conditions, while the acidic and more basic vitamins showed diverse retention behaviors. With the underivatized silica phase, increasing the salt concentration of the mobile phase resulted in enhanced retention of the acidic vitamins, but decreased retention of the basic vitamins. These observations thus signify the involvement of secondary mechanisms, such as electrostatic interaction in the retention of these analytes. Under optimized conditions, a baseline separation of all vitamins was achieved with excellent peak efficiency. In addition, the effects of water content in the sample on retention and peak efficiency were examined, with sample stacking effects observed when the injected sample contained a high amount of water.

  7. How to separate ionic liquids: use of hydrophilic interaction liquid chromatography and mixed mode phases.

    PubMed

    Lamouroux, C; Foglia, G; Le Rouzo, G

    2011-05-20

    This chromatographic study deals with the development of a convenient and versatile method to separate Room Temperature Ionic Liquids. Different modes of chromatography were studied. The study attempts to answer the following question: "what were the most important interactions for the separation of ionic liquids?". The results show that the essential interactions to assure a good retention of RTILs are the ionic ones and that hydrophobic interactions play a role in the selectivity of the separation. The separation of five imidazolium salt with a traditional diol columns in Hydrophilic Interaction Chromatography (HILIC) was demonstrated. It shows that neutral diol grafted column allows an important retention that we assume is due to the capability of diol to develop a thick layer of water. Furthermore, stationary phase based on mixed interaction associating ion exchange and hydrophobic properties were studied. Firstly, it will be argued that it is possible to separate RTILs with a convenient retention and resolution according to a reverse phase elution with the Primesep columns made of a brush type long alkyl chain with an embedded negatively charged functional group. Secondly, a sucessful separation of RTILs in HILIC mode with a mixed phase column containing a cationic exchanger and a hydrophobic octyl chain length will be demonstrated.

  8. Preparation and retention mechanism exploration of mesostructured cellular foam silica as stationary phase for high performance liquid chromatography.

    PubMed

    Sun, Shaoai; Zhang, Xiaoqiong; Han, Qiang; Wan, Wei; Ding, Mingyu

    2016-01-01

    Siliceous mesostructured cellular foam (MCF) with highly interconnected porous structure, ultralarge pore size and relatively uniform particle size (3-5μm) was prepared to achieve the mixed-mode and efficient separation of intact proteins. And molecular sieving effect for the first time played an important role in protein separation using mesoporous silica materials as HPLC stationary phase. The spherical silica particles were synthesized via hydrothermal method and the pore size was easily regulated by adding NH4F as well as altering the aging time. After aminopropyl derivatization, the chromatographic performance of functionalized mesoporous silica particles was investigated in comparison with those without modification and commercial NH2 column, and their mixed-mode retention mechanisms were investigated in detail. The superior separation performance for the retention of proteins was obtained on our home-made column in comparison with commercial NH2 column. The influences of aminopropyl derivatization and mobile phase composition on the column property were also investigated. Moreover, the home-made column showed similar performance for separation of polar anilines and neutral PAHs with the commercial column, owing to mixed-mode retention mechanisms including p-π stacking, electron interaction, hydrophobic effect, π-π EDA interaction and hydrogen bonding. All these results indicated that the aminopropyl modified MCF would be promising in the mixed-mode and efficient separation of biomolecules in addition with small molecules.

  9. Master equation approach for interacting slow- and stationary-light polaritons

    SciTech Connect

    Kiffner, M.; Hartmann, M. J.

    2010-09-15

    A master equation approach for the description of dark-state polaritons in coherently driven atomic media is presented. This technique provides a description of light-matter interactions under conditions of electromagnetically induced transparency (EIT) that is well suited for the treatment of polariton losses. The master equation approach allows us to describe general polariton-polariton interactions that may be conservative, dissipative, or a mixture of both. In particular, it enables us to study dissipation-induced correlations as a means for the creation of strongly correlated polariton systems. Our technique reveals a loss mechanism for stationary-light polaritons that has not been discussed so far. We find that polariton losses in level configurations with nondegenerate ground states can be a multiple of those in level schemes with degenerate ground states.

  10. Evaluation of hydrophilic interaction chromatography versus reversed-phase chromatography in a plant metabolomics perspective.

    PubMed

    T'kindt, Ruben; Storme, Michael; Deforce, Dieter; Van Bocxlaer, Jan

    2008-05-01

    The metabolomics goal, the unbiased relative quantification of all metabolites in a biological system, still lacks a universal analytical approach. In the LC-MS line of approach, one of the major problems encountered is the polar nature of a large group of (plant) metabolites. Here, we investigate the potential of hydrophilic interaction chromatography (HILIC) and compare its qualities with extended polarity RP chromatography. Two opposite LC phase compositions (Atlantis dC18 vs. TSKgel Amide-80) are compared in a plant metabolomics setting. Both performed equally well with regard to retentive capacities, but variation in peak area was about 5% higher for the HILIC approach. Focussing on matrix effects (ME) on the other hand, it was observed that this well-known problem in RP LC-MS metabolomics was not reduced on using hydrophilic interaction chromatography.

  11. Size-exclusion chromatography system for macromolecular interaction analysis

    DOEpatents

    Stevens, Fred J.

    1988-01-01

    A low pressure, microcomputer controlled system employing high performance liquid chromatography (HPLC) allows for precise analysis of the interaction of two reversibly associating macromolecules such as proteins. Since a macromolecular complex migrates faster than its components during size-exclusion chromatography, the difference between the elution profile of a mixture of two macromolecules and the summation of the elution profiles of the two components provides a quantifiable indication of the degree of molecular interaction. This delta profile is used to qualitatively reveal the presence or absence of significant interaction or to rank the relative degree of interaction in comparing samples and, in combination with a computer simulation, is further used to quantify the magnitude of the interaction in an arrangement wherein a microcomputer is coupled to analytical instrumentation in a novel manner.

  12. Pi-selective stationary phases: (III) Influence of the propyl phenyl ligand density on the aromatic and methylene selectivity of aromatic compounds in reversed phase liquid chromatography.

    PubMed

    Stevenson, Paul G; Soliven, Arianne; Dennis, Gary R; Gritti, Fabrice; Guiochon, Georges; Shalliker, R Andrew

    2010-08-13

    The retention characteristics of phenyl type stationary phases for reversed phase high performance liquid chromatography are still largely unknown. This paper explores the retention process of these types of stationary phases by examining the retention behaviour of linear PAHs and n-alkylbenzenes on a series of propyl phenyl stationary phases that have changes in their ligand density (1.23, 1.31, 1.97, 2.50 micromol m(-2)). The aromatic and methylene selectivities increased with increasing ligand density until a point where a plateau was observed, overall the propyl phenyl phases had a higher degree of aromatic selectivity than methylene selectivity indicating that these columns are suitable for separations involving aromatic compounds. Also, retention characteristics relating to the size of the solute molecule were observed to be influenced by the ligand density. It is likely that the changing retention characteristics are caused by the different topologies of the stationary phases at different ligand densities. At high ligand densities, the partition coefficient became constant. 2010 Elsevier B.V. All rights reserved.

  13. Identifying important structural features of ionic liquid stationary phases for the selective separation of nonpolar analytes by comprehensive two-dimensional gas chromatography.

    PubMed

    Zhang, Cheng; Ingram, Isaiah C; Hantao, Leandro W; Anderson, Jared L

    2015-03-20

    A series of dicationic ionic liquid (IL)-based stationary phases were evaluated as secondary columns in comprehensive two-dimensional gas chromatography (GC×GC) for the separation of aliphatic hydrocarbons from kerosene. In order to understand the role that structural features of ILs play on the selectivity of nonpolar analytes, the solvation parameter model was used to probe the solvation properties of the IL-based stationary phases. It was observed that room temperature ILs containing long free alkyl side chain substituents and long linker chains between the two cations possess less cohesive forces and exhibited the highest resolution of aliphatic hydrocarbons. The anion component of the IL did not contribute significantly to the overall separation, as similar selectivities toward aliphatic hydrocarbons were observed when examining ILs with identical cations and different anions. In an attempt to further examine the separation capabilities of the IL-based GC stationary phases, columns of the best performing stationary phases were prepared with higher film thickness and resulted in enhanced selectivity of aliphatic hydrocarbons. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Use and application of hydrophobic interaction chromatography for protein purification.

    PubMed

    McCue, Justin T

    2014-01-01

    The objective of this section is to provide the reader with guidelines and background on the use and experimental application of Hydrophobic Interaction chromatography (HIC) for the purification of proteins. The section will give step by step instructions on how to use HIC in the laboratory to purify proteins. General guidelines and relevant background information is also provided.

  15. Use of a biomimetic chromatographic stationary phase for study of the interactions occurring between inorganic anions and phosphatidylcholine membranes.

    PubMed Central

    Hu, Wenzhi; Haddad, Paul R; Hasebe, Kiyoshi; Mori, Masanobu; Tanaka, Kazuhiko; Ohno, Masako; Kamo, Naoki

    2002-01-01

    A liquid chromatographic method for the study of ion-membrane interactions is reported. A phosphatidylcholine biomimetic stationary phase was established by loading dimyristoylphosphatidylcholine (DMPC) onto a reversed-phase octadecylsilica packed column. This column was then used to study the interaction of some inorganic anions with the stationary phase by UV and conductivity detection. Ten inorganic anions were selected as model ions and were analyzed with the proposed chromatographic system. Anion-DMPC interactions of differing magnitudes were observed for all of the model anions. Perchlorate-DMPC interactions were strongest, followed by thiocyanate-DMPC, iodide-DMPC, chlorate-DMPC, nitrate-DMPC, bromide-DMPC, chloride-DMPC, fluoride-DMPC, and then sulfate-DMPC. Cations in the eluent, especially H(+) ions and divalent cations such as Ca(2+), showed strong effects on anion-DMPC interactions. The chromatographic data suggest that DMPC interacts with both the anions and the cations. Anion-DMPC interactions were dependent on the surface potential of the stationary phase: at low surface potentials anion-DMPC interactions were predominantly solvation dependent in nature whereas at more positive surface potentials anion-DMPC interactions were predominantly electrostatic in nature. Cation-DMPC interactions served to raise the surface potential, causing the anion-DMPC interactions to vary from solvation dependent to electrostatic. The chromatographic data were used to provide quantitative estimates of the enthalpies of the anion-DMPC interactions. PMID:12496102

  16. Going beyond the stationary flux towers to assess the interactions of land use and climate

    NASA Astrophysics Data System (ADS)

    Yakir, Dan; Rohatyn, shani; Ramati, Efrat; Tatrinov, Fedor; Rotenberg, Eyal

    2017-04-01

    Networks of permanent, stationary flux towers that allows continuous canopy-scale measurements over annual time-scales have revolutionized the study of the contemporary carbon cycle over the past two decades. However, this approach is limited in addressing questions related to dynamic changes in land use, vegetation types, disturbance, and their interactions with variations in environmental conditions. Using mobile laboratory for measuring CO2, water, energy, COS, and VOC fluxes, permitted us to extend our stationary flux tower measurements across many sites, but also limited measurements to short-time campaigns (days to weeks). To overcome this limitation, we adopted an empirical approach (often used in remote sensing) and used state of the art campaign-based ecosystem flux measurements to 'calibrate' local meteorological data available on continuous basis, to estimate annual-scale carbon, water, and energy budgets. Using this approach, we investigated the interactions of land use change (afforestation) and climate (humid Mediterranean to semi-arid, 730 to 300 mm in annual precipitation) on the ecosystem fluxes. The results showed that across this climatic range, afforestation increased ET markedly more in the wet (+200 mm yr-1 or 30% of P) than in the dry end (+58 mm yr-1 or 19% of P). Similarly, increase in carbon sequestration (NEE) associated with forestation was greater in the wet sites (+460 gC m-2 yr-1) than in the dry sites (+30 gC m-2 yr-1). In contrast, ecosystem net-radiation (Rn) and sensible heat flux (H) increased due to afforestation much more in the dry sites than in the wet sites ( 47 vs. 27 and 49 vs. 17 Wm-2, respectively). COS and VOC fluxes were also measured but reported separately. The results provided quantitative assessment of shifts in the tradeoffs associated with afforestation in this region, between the hydrological and energy-budget 'costs', vs. carbon sequestration and other ecosystem services, (e.g, surface cooling, erosion

  17. Protein self-interaction chromatography on a microchip.

    PubMed

    Deshpande, Kedar; Ahamed, Tangir; van der Wielen, Luuk A M; Horst, Joop H Ter; Jansens, Peter J; Ottens, Marcel

    2009-02-21

    This paper presents the development of a novel miniaturized experimental procedure for the measurement of protein-protein interactions through Self-Interaction Chromatography (SIC) on a microchip, without the use of chromatographic resins. SIC was recently demonstrated to be a relatively easy method to obtain quantitative thermodynamic information about protein-protein interactions, like the osmotic second virial coefficient B(22), which relates to protein phase behavior including protein crystallization. This successful miniaturization to microchip level of a measurement device for protein self-interaction data is a first key step to a complete microfluidic screening platform for the rational design of protein crystallizations, using substantially less expensive protein and experimentation time.

  18. Stationary Vortex Loops Induced by Filament Interaction and Local Pinning in a Chemical Reaction-Diffusion System

    NASA Astrophysics Data System (ADS)

    Jiménez, Zulma A.; Steinbock, Oliver

    2012-08-01

    Scroll rings are three-dimensional excitation waves rotating around one-dimensional filament loops. In experiments with the Belousov-Zhabotinsky reaction we show that the collapse of these loops can be stopped by local pinning to only two unexcitable heterogeneities. The resulting vortices rotate around stationary but curved filaments. The absence of filament motion can be explained by repulsive interaction that counteracts the expected curvature-induced motion. The shape and key dependencies of the stationary filaments are well described by a curvature-flow model with additive interaction velocities that rapidly decrease with filament distance.

  19. Phenyl Functionalized Sol-gel Silica Sorbent for Capillary Microextraction and Chromia-Based Sol-gel Ucon Stationary Phase for Capillary Gas Chromatography

    NASA Astrophysics Data System (ADS)

    McLean, Michael M.

    The first chapter of this thesis presents an introduction to sol-gel methodology whose usefulness as a synthetic route will be demonstrated with two applications in chromatography. The first application involves the fabrication of a capillary micro-extraction (CME) device by coating a phenyl functionalized extracting phase on the inner surface of a fused silica capillary for analyte pre-concentration. The device was coupled on-line to a RP-HPLC system and practicality was demonstrated using allergens as target analytes. The allergens chosen as model analytes are typically found in fragrance products and food. Most of the 26 fragrance allergens that are monitored by various government authorities have a phenyl organic moiety (a strong chromophore), thus making them appropriate probes for exploring the extraction efficiency of the coating using a UV detector. The CME device showed ppt level limit of detection which makes it suitable for trace analyses of allergens and similar compounds in a variety of matrices. The second application explores the feasibility of using sol-gel derived chromia-based stationary phase in gas chromatographic columns. The organic moiety of the stationary phase was derived from Ucon 75-H-90,000 while the inorganic backbone was prepared using chromium(III) dichloride hydroxide - methacrylic acid - aqua complex, 40% in isopropanol/acetone . Usefulness of prepared chromia-based GC stationary phase was examined for petrochemical application. Promising results were obtained using aliphatic-aromatics, polyaromatic hydrocarbons, BTEX test mixture, cycloalkanes, branched alkanes and akylbenzenes. The column was able to perform without degradation despite being rinsed multiples times sequentially with the following solvents: dichloromethane, methanol, water and finally methanol again. Maximum theoretical plate number calculated is around 2,400 plates/m. The plate number clearly needs improvement but is a promising result for the newly explored

  20. Correlations between the zeta potentials of silica hydride-based stationary phases, analyte retention behaviour and their ionic interaction descriptors.

    PubMed

    Kulsing, Chadin; Yang, Yuanzhong; Munera, Caesar; Tse, Colby; Matyska, Maria T; Pesek, Joseph J; Boysen, Reinhard I; Hearn, Milton T W

    2014-03-19

    In this study, the zeta potentials of type-B silica, bare silica hydride, the so-called Diamond Hydride™ and phenyl substituted silica hydride stationary phases have been measured in aqueous-organic media and correction procedures developed to account for the more negative zeta potential values in media containing different acetonitrile contents. Retention studies of 16 basic, acidic and neutral compounds were also performed with these four stationary phases with mobile phases containing 0.1% (v/v) formic acid and various acetonitrile-water compositions ranging from 0-90% (v/v) acetonitrile. The retention properties of these analytes were correlated to the corrected stationary phase zeta potentials measured under these different mobile phase conditions with R(2) values ranging from 0.01 to 1.00, depending on the stationary phase and analyte type. Using linear solvation energy relationships, stationary phase descriptors for each stationary phase have been developed for the different mobile phase conditions. Very high correlations of the zeta potentials with the ionic interaction descriptors were obtained for the type-B silica and the Diamond Hydride™ phases and good correlation with bare silica hydride material whilst there was no correlation observed for the phenyl substituted silica hydride phase. The nature of the retention mechanisms which gives rise to these different observations is discussed. The described methods represent a useful new approach to characterize and assess the retention properties of silica-hydride based chromatographic stationary phases of varying bonded-phase coverage and chemistries, as would be broadly applicable to other types of stationary phase used in the separation sciences. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Flow interaction between a streamwise oscillating cylinder and a downstream stationary cylinder

    NASA Astrophysics Data System (ADS)

    Xu, S. J.; Gan, L.; Zhou, Y.

    2016-11-01

    In this paper, we present some experimental results about the physical effects of a cylinder's streamwise oscillation motion on a downstream one in a tandem arrangement. The upstream cylinder undergoes a controlled simple harmonic oscillation at amplitudes A/ d = 0.2-0.8, where d is the cylinder diameter, and the frequency ratio of f_e/f_s = 0-3.0, where f_e is the cylinder oscillation frequency and f_s is the natural frequency of vortex shedding from a single stationary cylinder. Under these conditions, the vortex shedding is locked to the controlled oscillation motion. Flow visualisation using the planar laser-induced fluorescence and qualitative measurements using hot-wire anemometry reveal three distinct flow regimes behind the downstream cylinder. For f_e/f_s > (f_e/f_s)_c, where (f_e/f_s)_c is a critical frequency ratio which depends on A/ d and Reynolds number Re, a so-called SA-mode occurs. The upstream oscillating cylinder generates binary vortices symmetrically arranged about the centreline, each containing a pair of counter-rotating vortices, and the downstream cylinder sheds vortices alternately at 0.5f_e. For 0.7-1.0 < f_e/f_s < (f_e/f_s)_c a complex vortex street that consists of two outer rows of vortices generated by the oscillating cylinder and two inner rows of vortices shed from the downstream stationary cylinder, which is referred to as AA-mode. For 0.3-0.6 < f_e/f_s< 0.8-1.0, one single staggered vortex street (A-mode) is observed. It is also found that, when f_e/f_s is near unity, the streamwise interaction of the two cylinders gives rise to the most energetic wake in the cross-stream direction, in terms of its maximum width, and the wake is AA-mode-like. The effects of other parameters such as the spacing between the two cylinders, Re and A/ d on the flow pattern are also discussed in details. The observations are further compared to the stationary tandem cylinder cases.

  2. Two-dimensional high-performance thin-layer chromatography of tryptic bovine albumin digest using normal- and reverse-phase systems with silanized silica stationary phase.

    PubMed

    Gwarda, Radosław Łukasz; Dzido, Tadeusz Henryk

    2013-10-18

    Among many advantages of planar techniques, two-dimensional (2D) separation seems to be the most important for analysis of complex samples. Here we present quick, simple and efficient two-dimensional high-performance thin-layer chromatography (2D HPTLC) of bovine albumin digest using commercial HPTLC RP-18W plates (silica based stationary phase with chemically bonded octadecyl ligands of coverage density 0.5μmol/m(2) from Merck, Darmstadt). We show, that at low or high concentration of water in the mobile phase comprised methanol and some additives the chromatographic systems with the plates mentioned demonstrate normal- or reversed-phase liquid chromatography properties, respectively, for separation of peptides obtained. These two systems show quite different separation selectivity and their combination into 2D HPTLC process provides excellent separation of peptides of the bovine albumin digest. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  4. Adsorption of water from aqueous acetonitrile on silica-based stationary phases in aqueous normal-phase liquid chromatography.

    PubMed

    Soukup, Jan; Jandera, Pavel

    2014-12-29

    Excess adsorption of water from aqueous acetonitrile mobile phases was investigated on 16 stationary phases using the frontal analysis method and coulometric Karl-Fischer titration. The stationary phases include silica gel and silica-bonded phases with different polarities, octadecyl and cholesterol, phenyl, nitrile, pentafluorophenylpropyl, diol and zwitterionic sulfobetaine and phosphorylcholine ligands bonded on silica, hybrid organic-silica and hydrosilated matrices. Both fully porous and core-shell column types were included. Preferential uptake of water by the columns can be described by Langmuir isotherms. Even though a diffuse rather than a compact adsorbed discrete layer of water on the adsorbent surface can be formed because of the unlimited miscibility of water with acetonitrile, for convenience, the preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water strongly depends on the polarity and type of the column. Less than one monomolecular water layer equivalent was adsorbed on moderate polar silica hydride-based stationary phases, Ascentis Express F5 and Ascentis Express CN column at the saturation capacity, while on more polar stationary phases, several water layer equivalents were up-taken from the mobile phase. The strongest affinity to water was observed on the ZIC cHILIC stationary phases, where more than nine water layer equivalents were adsorbed onto its surface at its saturation capacity. Columns with bonded hydroxyl and diol ligands show stronger water adsorption in comparison to bare silica. Columns based on hydrosilated silica generally show significantly decreased water uptake in comparison to stationary phases bonded on ordinary silica. Significant correlations were found between the water uptake and the separation selectivity for compounds with strong polarity differences.

  5. Interaction of zones of flow separation in a centrifugal impeller-stationary vane system

    NASA Astrophysics Data System (ADS)

    Akin, O.; Rockwell, D.

    1994-10-01

    In a radial flow pump operating in off-design conditions, regions of stall can exist on the rotating impeller blade and on the downstream diffuser blade, vane or tongue. Interaction of these stall zones can generate complex patterns of vorticity concentrations. In turn, these vorticity concentrations are related to sources of unsteady stagnation enthalpy. The form of these patterns is strongly dependent on the instantaneous location of the impeller trailing-edge relative to the leading-edge of the vane. Comparison of instantaneous with ensemble-averaged images shows that the flow structure in the gap region between the impeller and the vane is highly repetitive. Away from this region, in particular in the separated shear layer from the vane, the nonrepetitive nature of the vorticity field is manifested in substantial reduction of peak levels of vorticity in the ensemble-averaged image, relative to the instantaneous image. The three-dimensional flow structure resulting from these separation zone interactions was characterized via end views of the flow patterns. Particularly pronounced concentrations of vorticity can occur in this plane. They tend to be located in the shear layer at the outer edge of the large-scale separation zone. These vorticity concentrations are, however, highly non-stationary for successive passages of the impeller blade. Ensemble-averaging reveals that they persist primarily on the endwalls of the diffuser.

  6. Comparison of three development approaches for Stationary Phase Optimised Selectivity Liquid Chromatography based screening methods Part II: A group of structural analogues (PDE-5 inhibitors in food supplements).

    PubMed

    Deconinck, E; Ghijs, L; Kamugisha, A; Courselle, P

    2016-02-01

    Three approaches for the development of a screening method to detect adulterated dietary supplements, based on Stationary Phase Optimised Selectivity Liquid Chromatography were compared for their easiness/speed of development and the performance of the optimal method obtained. This comparison was performed for a heterogeneous group of molecules, i.e. slimming agents (Part I) and a group of structural analogues, i.e. PDE-5 inhibitors (Part II). The first approach makes use of primary runs at one isocratic level, the second of primary runs in gradient mode and the third of primary runs at three isocratic levels to calculate the optimal combination of segments of stationary phases. In each approach the selection of the stationary phase was followed by a gradient optimisation. For the PDE-5 inhibitors, the group of structural analogues, only the method obtained with the third approach was able to differentiate between all the molecules in the development set. Although not all molecules are baseline separated, the method allows the identification of the selected adulterants in dietary supplements using only diode array detection. Though, due to the mobile phases used, the method could also be coupled to mass spectrometry. The method was validated for its selectivity following the guidelines as described for the screening of pesticide residues and residues of veterinary medicines in food.

  7. Application of polymethacrylate resin as stationary phase in liquid chromatography with UV detection for C1-C7 aliphatic monocarboxylic acids and C1-C7 aliphatic monoamines.

    PubMed

    Ohta, Kazutoku; Towata, Atsuya; Ohashi, Masayoshi; Takeuchi, Toyohide

    2004-06-11

    The application of unfunctionized polymethacrylate resin (TSKgel G3000PWXL) as a stationary phase in liquid chromatography with UV detection for C1-C7 aliphatic monocarboxylic acids (formic acid, acetic acid, propionic acid, butyric acid, isovaleric acid, valeric acid, 3,3-dimethylbutyric acid, 4-methylvaleric acid, hexanoic acid, 2-methylhexanoic acid, 5-methylhexanoic acid and heptanoic acid) and C1-C7 aliphatic monoamines (methylamine, ethylamine, propylamine, isobutylamine, butylamine, isoamylamine, amylamine, 1,3-dimethylbutylamine, hexylamine, 2-heptylamine and heptylamine) was carried out. Using dilute sulfuric acid as the eluent, the TSKgel G3000PWXL, resin acted as an advanced stationary phase for these C1-C7 carboxylic acids. Excellent simultaneous separation and symmetrical peaks for these C1-C7 carboxylic acids were achieved on a TSKgel G3000PWXL column (150 mm x 6 mm i.d.) in 60 min with 0.25 mM sulfuric acid containing 1 mM 2-methylheptanoic acid at pH 3.3 as the eluent. Using dilute sodium hydroxide as the eluent, the TSKgel G3000PWXL resin also behaved as an advanced stationary phase for these C1-C7 amines. Excellent simultaneous separation and good peaks for these C1-C7 amines were achieved on the TSKgel G3000PWXL column in 60 min with 10 mM sodium hydroxide containing 0.5 mM 1-methylheptylamine at pH 11.9 as the eluent.

  8. Construction of a new hydrazone-linked chiral covalent organic framework-silica composite as the stationary phase for high performance liquid chromatography.

    PubMed

    Zhang, Kai; Cai, Song-Liang; Yan, Yi-Lun; He, Zi-Hao; Lin, Hui-Mei; Huang, Xiao-Ling; Zheng, Sheng-Run; Fan, Jun; Zhang, Wei-Guang

    2017-09-05

    Covalent organic frameworks (COFs), as an emerging class of crystalline porous organic polymers, have great potential for applications in chromatographic separation owning to their fascinating crystalline structures and outstanding properties. However, development of COF materials as novel stationary phases in high performance liquid chromatography (HPLC) is just in its infancy. Herein, we report the design and construction of a new hydrazone-linked chiral COF, termed BtaMth COF, from a chiral hydrazide building block (Mth) and present a one-pot synthetic method for the fabrication of BtaMth@SiO2 composite for HPLC separation of isomers. The as-synthesized BtaMth chiral COF displays good crystallinity, high porosity, as well as excellent chemical stability. Meanwhile, the fabricated HPLC column by using BtaMth@SiO2 composite as the new stationary phase exhibits high resolution performances for the separation of positional isomers including nitrotoluene and nitrochlorobenzene, as well as cis-trans isomers including beta-cypermethrin and metconazole. Additionally, some effects such as the composition of the mobile phase and column temperature for HPLC separations on the BtaMth@SiO2 packed column also have been studied in detail. The successful applications indicate the great potentials of hydrazone-linked chiral COF-silica composite as novel stationary phase for the efficient HPLC separation. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Determination of volatile compounds in cider apple juices using a covalently bonded ionic liquid coating as the stationary phase in gas chromatography.

    PubMed

    Pello-Palma, Jairo; González-Álvarez, Jaime; Gutiérrez-Álvarez, María Dolores; Dapena de la Fuente, Enrique; Mangas-Alonso, Juan José; Méndez-Sánchez, Daniel; Gotor-Fernández, Vicente; Arias-Abrodo, Pilar

    2017-04-01

    A chromatographic method for the separation of volatile compounds in Asturian cider apple juices has been developed. For this separation purpose, a monocationic imidazolium-based ionic liquid bearing a reactive terminal iodine atom was synthesized by a quaternization-anion exchange chemical sequence. Next, the gas chromatography (GC) stationary phase was prepared by covalently linking the imidazolium monolith to the reactive silanol groups of the inner capillary wall at 70 °C. This coated GC column exhibited good thermal stability (290 °C), as well as good efficiency (2000 plates/m) in the separation of volatile compounds from Asturian apple cider juices, and was characterized using the Abraham solvation parameter model. The intra-day and inter-day precision of the chromatographic method was evaluated, obtaining relative standard deviations from 3.7 to 12.9% and from 7.4 to 18.0%, respectively. Furthermore, recoveries from 82.5 to 122% were achieved. Graphical Abstract Covalent bonding of an ionic liquid to inner column wall led to a great improvement of the separation efficiencies of stationary phases in gas chromatography.

  10. Simultaneous analysis of multiple neurotransmitters by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry.

    PubMed

    Tufi, Sara; Lamoree, Marja; de Boer, Jacob; Leonards, Pim

    2015-05-22

    Neurotransmitters are endogenous metabolites that allow the signal transmission across neuronal synapses. Their biological role is crucial for many physiological functions and their levels can be changed by several diseases. Because of their high polarity, hydrophilic interaction liquid chromatography (HILIC) is a promising tool for neurotransmitter analysis. Due to the large number of HILIC stationary phases available, an evaluation of the column performances and retention behaviors has been performed on five different commercial HILIC packing materials (silica, amino, amide and two zwitterionic stationary phases). Several parameters like the linear correlation between retention and the distribution coefficient (logD), the separation factor k and the column resolution Rs have been investigated and the column performances have been visualized with a heat map and hierarchical clustering analysis. An optimized and validated HILIC-MS/MS method based on the ZIC-cHILIC column is proposed for the simultaneous detection and quantification of twenty compounds consisting of neurotransmitters, precursors and metabolites: 3-methoxytyramine (3-MT), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxy-L-tripthophan, acetylcholine, choline, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, epinephrine, γ-aminobutyric acid (GABA), glutamate, glutamine, histamine, histidine, L-tryptophan, L-tyrosine, norepinephrine, normetanephrine, phenylalanine, serotonin and tyramine. The method was applied to neuronal metabolite profiling of the central nervous system of the freshwater snail Lymnaea stagnalis. This method is suitable to explore neuronal metabolism and its alteration in different biological matrices.

  11. [Linear and nonlinear spectroscopic probing of solute interactions with chemically modified silica surface]. [Stationary phase studies

    SciTech Connect

    Not Available

    1993-01-01

    Objective is to understand the surface science underlying liquid chromatographic separations, enabling improvements in design of chromatographic stationary phases. Progress was made both in use of laser spectroscopy to probe chromatographic surfaces and in developing new stationary phases based on self-assembled monolayers.

  12. Chromatography.

    ERIC Educational Resources Information Center

    Brantley, L. Reed, Sr.; Demanche, Edna L.; Klemm, E. Barbara; Kyselka, Will; Phillips, Edwin A.; Pottenger, Francis M.; Yamamoto, Karen N.; Young, Donald B.

    This booklet presents some activities on chromatography. Directions for preparing leaf pigment extracts using alcohol are given, and paper chromatography and thin-layer chromatography are described as modifications of the basic principles of chromatography. (KHR)

  13. Chromatography.

    ERIC Educational Resources Information Center

    Brantley, L. Reed, Sr.; Demanche, Edna L.; Klemm, E. Barbara; Kyselka, Will; Phillips, Edwin A.; Pottenger, Francis M.; Yamamoto, Karen N.; Young, Donald B.

    This booklet presents some activities on chromatography. Directions for preparing leaf pigment extracts using alcohol are given, and paper chromatography and thin-layer chromatography are described as modifications of the basic principles of chromatography. (KHR)

  14. Assessment of intra-particle diffusion in hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography under conditions of identical packing structure.

    PubMed

    Song, Huiying; Desmet, Gert; Cabooter, Deirdre

    2017-06-29

    A recently developed stripping protocol to completely remove the stationary phase of reversed-phase liquid chromatography (RPLC) columns and turn them into hydrophilic interaction liquid chromatography (HILIC) columns with identical packing characteristics is used to study the underlying mechanisms of intra-particle diffusion in RPLC and HILIC. The protocol is applied to a column with a large geometrical volume (250×4.6mm, 5μm) to avoid extra-column effects and for compounds with a broad range in retention factors (k" from ∼0.6 to 8). Three types of behavior for the intra-particle diffusion (Dpart/Dm) in RPLC versus HILIC can be distinguished: for nearly unretained compounds (k"<0.6), intra-particle diffusion in HILIC is larger than in RPLC; for compounds with intermediate retention behavior (k"∼0.9-1.2), intra-particle diffusion in HILIC and RPLC are similar; and for well retained compounds (k">1.8), intra-particle diffusion in RPLC is larger than in HILIC. To explain these observations, diffusion in the stationary phase (γsDs) and in the stagnant mobile phase in the mesopore zone (γmpDm) are deduced from experimentally determined values of the intra-particle diffusion, using models derived from the Effective Medium Theory. It is demonstrated that the larger intra-particle diffusion obtained for slightly retained compounds under HILIC conditions is caused by the higher mesopore diffusion in HILIC (γmp=0.474 for HILIC versus 0.435 for RPLC), while the larger intra-particle diffusion obtained for strongly retained compounds under RPLC conditions can be related to the much higher stationary phase diffusion in RPLC (γsDs/Dm=0.200 for RPLC versus 0.113 for HILIC). Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Protein interactions in hydrophobic charge induction chromatography (HCIC).

    PubMed

    Ghose, Sanchayita; Hubbard, Brian; Cramer, Steven M

    2005-01-01

    A quantitative understanding of how proteins interact with hydrophobic charge induction chromatographic resins is provided. Selectivity on this mode of chromatography for monoclonal antibodies as compared to other model proteins is probed by means of a linear retention vs pH plot. The pH-dependent adsorption behavior on this mode of chromatography for a hydrophobic, charged solute is described by taking into account the equilibrium between a hydrophobic, charged solute and an ionizable, heterocyclic ligand. By analogy, an equation that is seen to adequately describe macromolecular retention under linear conditions over a range of pH is developed. A preparative, nonlinear isotherm that can capture both pH and salt concentration dependency for proteins is proposed by using an exponentially modified Langmuir isotherm model. This model is seen to successfully simulate adsorption isotherms for a variety of proteins over a range of pHs and mobile phase salt concentrations. Finally, the widely differing retention characteristics of two monoclonal antibodies are used to derive two different strategies for improving separations on this mode of chromatography. A better understanding of protein binding to this class of resins is seen as an important step to future exploitation of this mode of chromatography for industrial scale purification of proteins.

  16. Preparation of hydrophilic polymer-grafted polystyrene beads for hydrophilic interaction chromatography via surface-initiated atom transfer radical polymerization.

    PubMed

    Dai, Xiaojun; He, Yuan; Wei, Yinmao; Gong, Bolin

    2011-11-01

    A one-step procedure based on surface-initiated atom transfer radical polymerization (SI-ATRP) to hydrophilize monodisperse poly(chloromethylstyrene-co-divinylbenzene) beads has been presented in this work, using 2-hydroxyl-3-[4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]propyl 2-methylacrylate (HTMA) as a monomer. The chain length of the grafted poly(HTMA) was controlled via varying the ratio of HTMA to initiator on the surface of the beads. When using the grafted beads as a stationary phase in hydrophilic interaction chromatography (HILIC), good resolution for nucleobases/nucleosides was obtained with acetonitrile aqueous solution as an eluent; while for phenolic acids and glycosides, they could be eluted and separated in the presence of TFA. The retention time of the solutes increased with the amount of the grafted HTMA. The retention mechanisms of solutes were investigated by the effects of mobile phase composition and buffer pH on the retention of solutes. The results illustrated that the retention behaviors of the tested solutes were dominated by hydrogen bonding interaction and electrostatic interaction. From the chemical structure of the ligands, the modified beads could not only be used as a stationary phase in HILIC, but also act as a useful building block to develop new stationary phases for other chromatographic modes such as affinity media. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Organic monoliths for hydrophilic interaction electrochromatography/chromatography and immunoaffinity chromatography

    PubMed Central

    Gunasena, Dilani N.; El Rassi, Ziad

    2012-01-01

    This article is aimed at providing a review of the progress made over the past decade in the preparation of polar monoliths for hydrophilic interaction liquid chromatography (HILIC)/capillary electrochromatography (HI-CEC) and in the design of immuno-monoliths for immunoaffinity chromatography (IAC) that are based on some of the polar monolith precursors used in HILIC/HI-CEC. In addition, this review article discusses some of the applications of polar monoliths by HILIC and HI-CEC, and the applications of immuno-monoliths. This article is by no means an exhaustive review of the literature; it is rather a survey of the recent progress made in the field with 83 references published in the past decade on the topics of HILIC and IAC monoliths. PMID:22147366

  18. Analysis of normal and modified nucleosides in urine samples by high-performance liquid chromatography with different stationary phases.

    PubMed

    Studzińska, S; Buszewski, Bogusław

    2014-08-01

    The main aim of the present work was to study the retention behavior and quantification of nine nucleosides with the use of octadecyl, alkylamide, cholesterol and alkyl-phosphate stationary phases. The influence of organic solvent and buffer concentration on the separation of these compounds was under investigation. The retention factor had the highest values for the octadecyl and cholesterol packing materials. Complete separation of all the studied nucleosides was achieved in case of cholesterol stationary phase. The optimized separation method was applied for the quantification of nucleosides in the urine samples. Calibration plots showed good linearity (R(2) > 0.999) and the limits of detection were in a range of 0.3-0.5 µg/mL, while the limits of quantitation were >0.9 µg/mL. Accuracy was in the range of 5-11%. Copyright © 2014 John Wiley & Sons, Ltd.

  19. Development of Hydrophilic Interaction Liquid Chromatography Method for the Analysis of Moxonidine and Its Impurities.

    PubMed

    Filipic, Slavica; Elek, Milica; Popović, Marija; Nikolic, Katarina; Agbaba, Danica

    2016-01-01

    Fast and simple hydrophilic interaction liquid chromatography (HILIC) method was developed and validated for the analysis of moxonidine and its four impurities (A, B, C, and D) in pharmaceutical dosage form. All experiments were performed on the Agilent Technologies 1200 high-performance liquid chromatography (HPLC) system using Zorbax RX-SIL, 250 mm × 4.6 mm, 5 μm column as stationary phase (T = 25°C, F = 1 mL/min, and λ = 255 nm), and mixture of acetonitrile and 40 mM ammonium formate buffer (pH 2.8) 80 : 20 (v/v) as mobile phase. Under the optimal chromatographic conditions, selected by central composite design, separation and analysis of moxonidine and its four impurities are enabled within 12 minutes. Validation of the method was conducted in accordance with ICH guidelines. Based on the obtained results selectivity, linearity (r ≥ 0.9976), accuracy (recovery: 93.66%-114.08%), precision (RSD: 0.56%-2.55%), and robustness of the method were confirmed. The obtained values of the limit of detection and quantification revealed that the method can be used for determination of impurities levels below 0.1%. Validated method was applied for determination of moxonidine and its impurities in commercially available tablet formulation. Obtained results confirmed that validated method is fast, simple, and reliable for analysis of moxonidine and its impurities in tablets.

  20. Development of Hydrophilic Interaction Liquid Chromatography Method for the Analysis of Moxonidine and Its Impurities

    PubMed Central

    2016-01-01

    Fast and simple hydrophilic interaction liquid chromatography (HILIC) method was developed and validated for the analysis of moxonidine and its four impurities (A, B, C, and D) in pharmaceutical dosage form. All experiments were performed on the Agilent Technologies 1200 high-performance liquid chromatography (HPLC) system using Zorbax RX-SIL, 250 mm × 4.6 mm, 5 μm column as stationary phase (T = 25°C, F = 1 mL/min, and λ = 255 nm), and mixture of acetonitrile and 40 mM ammonium formate buffer (pH 2.8) 80 : 20 (v/v) as mobile phase. Under the optimal chromatographic conditions, selected by central composite design, separation and analysis of moxonidine and its four impurities are enabled within 12 minutes. Validation of the method was conducted in accordance with ICH guidelines. Based on the obtained results selectivity, linearity (r ≥ 0.9976), accuracy (recovery: 93.66%–114.08%), precision (RSD: 0.56%–2.55%), and robustness of the method were confirmed. The obtained values of the limit of detection and quantification revealed that the method can be used for determination of impurities levels below 0.1%. Validated method was applied for determination of moxonidine and its impurities in commercially available tablet formulation. Obtained results confirmed that validated method is fast, simple, and reliable for analysis of moxonidine and its impurities in tablets. PMID:27847672

  1. Determination of molindone enantiomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry using macrocyclic antibiotic chiral stationary phases.

    PubMed

    Jiang, Hongliang; Li, Yinghe; Pelzer, Mary; Cannon, Michelle J; Randlett, Christopher; Junga, Heiko; Jiang, Xiangyu; Ji, Qin C

    2008-05-30

    A sensitive and selective bioanalytical assay was developed and validated for the determination of enantiomeric molindone in human plasma using high-performance liquid chromatography-tandem mass spectrometry along with supported liquid extraction procedures. The chiral separation was evaluated and optimized on macrocyclic antibiotic type chiral stationary phases (CSPs) based on teicoplanin aglycone (Chirobiotic TAG) in polar organic, polar ionic, and reversed-phase mode chromatography, respectively. Complete baseline separation was achieved on a Chirobiotic TAG column under isocratic condition in reversed-phase chromatography. The method validation was conducted using a Chirobiotic TAG column (100 mm x 2.1 mm) over the curve range 0.100-100 ng/ml for each molindone enantiomer using 0.0500 ml of plasma sample. The flow rate was 0.8 ml/min and the total run time was 9 min. Supported liquid extraction in a 96-well plate format was used for sample preparation. Parameters including recovery, matrix effect, linearity, sensitivity, specificity, carryover, precision, accuracy, dilution integrity, and stability were evaluated. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels were RSD

  2. Green chromatography separation of analytes of greatly differing properties using a polyethylene glycol stationary phase and a low-toxic water-based mobile phase.

    PubMed

    Šatínský, Dalibor; Brabcová, Ivana; Maroušková, Alena; Chocholouš, Petr; Solich, Petr

    2013-07-01

    A simple, rapid, and environmentally friendly HPLC method was developed and validated for the separation of four compounds (4-aminophenol, caffeine, paracetamol, and propyphenazone) with different chemical properties. A "green" mobile phase, employing water as the major eluent, was proposed and applied to the separation of analytes with different polarity on polyethylene glycol (PEG) stationary phase. The chromatography separation of all compounds and internal standard benzoic acid was performed using isocratic elution with a low-toxicity mobile phase consisting of 0.04% (v/v) triethylamine and water. HPLC separation was carried out using a PEG reversed-phase stationary phase Supelco Discovery HS PEG column (15 × 4 mm; particle size 3 μm) at a temperature of 30 °C and flow rate at 1.0 mL min(-1). The UV detector was set at 210 nm. In this study, a PEG stationary phase was shown to be suitable for the efficient isocratic separation of compounds that differ widely in hydrophobicity and acid-base properties, particularly 4-aminophenol (log P, 0.30), caffeine (log P, -0.25), and propyphenazone (log P, 2.27). A polar PEG stationary phase provided specific selectivity which allowed traditional chromatographic problems related to the separation of analytes with different polarities to be solved. The retention properties of the group of structurally similar substances (aromatic amines, phenolic compounds, and xanthine derivatives) were tested with different mobile phases. The proposed green chromatography method was successfully applied to the analysis of active substances and one degradation impurity (4-aminophenol) in commercial preparation. Under the optimum chromatographic conditions, standard calibration was carried out with good linearity correlation coefficients for all compounds in the range (0.99914-0.99997, n = 6) between the peak areas and concentration of compounds. Recovery of the sample preparation was in the range 100 ± 5% for all compounds

  3. High-performance liquid chromatography of some basic drugs on a n-octadecylphosphonic acid modified magnesia-zirconia stationary phase.

    PubMed

    He, Hai-Bo; Feng, Yu-Qi; Da, Shi-Lu; Hu, Zhi-Xiong; Qu, Li

    2005-08-01

    The high-performance liquid chromatographic behavior of some basic drugs was studied on a n-octadecylphosphonic acid modified magnesia-zirconia (C18PZM) stationary phase. The effect of mobile phase variables such as methanol content, ionic strength, and pH on their chromatographic behavior was investigated. The retention mechanism of basic drugs on the stationary phase was elucidated. The results indicate that both hydrophobic and cation-exchange interactions contribute to solute retention under most chromatographic conditions. The inherent Brönsted-acid sites and also the adsorbed Lewis base anionic buffer constituents on accessible ZM surface Lewis acid sites play a role in the retention of ionized solutes by cation-exchange interaction. However, especially at high mobile phase pH, the retention of basic drugs depends mainly on hydrophobic interactions between solutes and support. Separations of the basic drugs on the C18PZM phase by a predominantly reversed-phase retention mode were very promising. The mixed-mode retention feature on this phase, as a result of the adsorbed Lewis base anionic buffer constituents acting as sites for cation-exchange, could also be very useful, e.g. for enhancing the chromatographic selectivity of such analytes. The C18PZM seems to be an excellent alternative to silica-based reversed-phase stationary phase for the separation of strongly basic solutes.

  4. Fractionation and recovery of whey proteins by hydrophobic interaction chromatography.

    PubMed

    Santos, Maria João; Teixeira, José A; Rodrigues, Lígia R

    2011-03-01

    A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.

  5. Stationary-point approach to the phase transition of the classical XY chain with power-law interactions.

    PubMed

    Kastner, Michael

    2011-03-01

    The stationary points of the potential energy function V of the classical XY chain with power-law pair interactions (i.e., interactions decaying like r{-α} with the distance) are analyzed. For a class of "spinwave-type" stationary points, the asymptotic behavior of the Hessian determinant of V is computed analytically in the limit of large system size. The computation is based on the Toeplitz property of the Hessian and makes use of a Szegö-type theorem. The results serve to illustrate a recently discovered relation between phase transitions and the properties of stationary points of classical many-body potentials. In agreement with this relation, the exact phase transition potential energy of the model can be read off from the behavior of the Hessian determinant for exponents α between zero and one. For α between one and two, the phase transition is not manifest in the behavior of the determinant, and it might be necessary to consider larger classes of stationary points.

  6. Monolayer-Protected Gold Nanoparticles as an Efficient Stationary Phase for Open Tubular Gas Chromatography using a Square Capillary Model for Chip-Based Gas Chromatography in Square Cornered Microfabricated Channels

    SciTech Connect

    Gross, Gwen M.; Grate, Jay W. ); Synovec, Robert E.

    2004-03-12

    The application of a dodecanethiol monolayer protected gold nanoparticle (MPN) stationary phase within a microchannel environment was explored using a square capillary column as a model for a high-speed, microfabricated gas chromatography (?GC). Successful deposition and evaluation of a dodecanethiol MPN phase within a 1.3 m long, 100?m by 100?m square capillary is reported. Depth of the MPN phase was evaluated using SEM analysis. An average thickness of 15 nm along the capillary walls was determined. While the film depth along the walls was very uniform, the corner depths were greater with the largest observed depth being 430 nm. Overall, an efficient chromatographic system was obtained with a minimum reduced plate height, hmin, of 1.2 for octane (k= 0.22). Characterization of the MPN column was completed using four compound classes (alkanes, alcohols, ketones, and aromatics) that were used to form a 7 component mixture with a 2 second separation. A mixture consisting of a nerve agent simulator in a sample containing analytes that may commonly interfere with detection was also separated in 2 seconds, much faster than a similar separation previously reported using a?GC system in 50 seconds. Application of the square capillary MPN column for a high-speed separation as the second column of a comprehensive two-dimensional gas chromatography system (GC x GC) was also explored. Comparison of the MPN stationary phase was compared to phases employed in previously reported?GC systems.

  7. High-throughput protein precipitation and hydrophobic interaction chromatography: salt effects and thermodynamic interrelation.

    PubMed

    Nfor, Beckley K; Hylkema, Nienke N; Wiedhaup, Koenraad R; Verhaert, Peter D E M; van der Wielen, Luuk A M; Ottens, Marcel

    2011-12-09

    Salt-induced protein precipitation and hydrophobic interaction chromatography (HIC) are two widely used methods for protein purification. In this study, salt effects in protein precipitation and HIC were investigated for a broad combination of proteins, salts and HIC resins. Interrelation between the critical thermodynamic salting out parameters in both techniques was equally investigated. Protein precipitation data were obtained by a high-throughput technique employing 96-well microtitre plates and robotic liquid handling technology. For the same protein-salt combinations, isocratic HIC experiments were performed using two or three different commercially available stationary phases-Phenyl Sepharose low sub, Butyl Sepharose and Resource Phenyl. In general, similar salt effects and deviations from the lyotropic series were observed in both separation methods, for example, the reverse Hofmeister effect reported for lysozyme below its isoelectric point and at low salt concentrations. The salting out constant could be expressed in terms of the preferential interaction parameter in protein precipitation, showing that the former is, in effect, the net result of preferential interaction of a protein with water molecules and salt ions in its vicinity. However, no general quantitative interrelation was found between salting out parameters or the number of released water molecules in protein precipitation and HIC. In other words, protein solubility and HIC retention factor could not be quantitatively interrelated, although for some proteins, regular trends were observed across the different resins and salt types.

  8. Development of a Stationary Phase Optimised Selectivity Liquid Chromatography based screening method for adulterations of food supplements for the treatment of pain.

    PubMed

    Deconinck, E; Kamugisha, A; Van Campenhout, P; Courselle, P; De Beer, J O

    2015-06-01

    Illegally adulterated dietary supplements are an increasing problem worldwide. One of the important groups of often adulterated products are the dietary supplements, sold for the treatment of pain. These often contain analgesics, a heterogeneous group of molecules, containing both hydrophilic and hydrophobic compounds. The development of a screening method for these components, especially when mass spectrometric detection is not available, necessitates chromatographic separation, difficult to achieve with traditional chromatographic columns. In this paper Stationary Phase Optimised Selectivity Liquid Chromatography was used for the development of a screening method for nine analgesics, codeine and caffeine, often present in this type of dietary supplements. The method shows a good separation of all the compounds, allowing the screening to be performed with diode array detection and is fully compatible with mass spectrometry. The method was validated for its selectivity following the guidelines as described for the screening of pesticide residues and residues of veterinary medicines in food.

  9. Development of LC chiral methods for neutral pharmaceutical related compounds using reversed phase and normal phase liquid chromatography with different types of polysaccharide stationary phases.

    PubMed

    Zhou, Lili; Welch, Chris; Lee, Clair; Gong, Xiaoyi; Antonucci, Vincent; Ge, Zhihong

    2009-05-01

    The enantioselectivity of a collection of neutral pharmaceutical compounds on six different types of polysaccharide chiral stationary phases (CSPs), Chiralpak AD (and AD-RH), Chiralcel OD (and OD-RH), Chiralpak OJ (and OJ-R), Chiralcel AS (and AS-RH), Sepapak-2 and Sepapak-4 are compared using reversed phase (RPLC) and normal phase liquid chromatography (NPLC). Screening strategies for maximizing the probability of achieving an initial chiral separation hit for neutral compounds using both RPLC and NPLC are described. Further method optimizations are demonstrated by modifying parameters such as organic modifier composition, eluent pH or CSP particle size. Several practical examples of the application of chiral methods for the study of synthetic reaction mixtures are presented. The most critical validation aspects, including limit of detection, specificity, and ruggedness, are also briefly presented.

  10. Preparation of a polybutadiene stationary phase immobilized by gamma radiation for reversed-phase high-performance liquid chromatography.

    PubMed

    Lopes, Nilva P; Collins, Kenneth E; Jardim, Isabel C S F

    2003-02-14

    Polybutadiene (PBD) has been immobilized on HPLC silica by gamma radiation doses in the range from 5 to 180 kGy. Columns prepared from these reversed-phase materials, as well as from similar non-irradiated materials, were tested with standard sample mixtures and characterized by elemental analysis (% C) and infrared spectroscopy. A low dose of 5 kGy is sufficient to produce a layer of immobilized PBD which functions as an efficient and stable stationary phase. Higher doses give thicker immobilized layers having less favorable chromatographic properties.

  11. Characterization of capillary-channeled polymer fiber stationary phases for high-performance liquid chromatography protein separations: Comparative analysis with a packed-bed column.

    PubMed

    Nelson, Dwella M; Marcus, R Kenneth

    2006-12-15

    Capillary-channeled polymer (C-CP) fibers are investigated as reversed-phase (RP) stationary phases for high-performance liquid chromatography of proteins. A comparative analysis of column characteristics for polypropylene and poly(ethylene terephthalate) C-CP fiber columns and a conventional packed-bed (C4-derivatized silica) column has been undertaken. Five proteins (ribonuclease A, cytochrome c, lysozyme, myoglobin, bovine serum albumin) were used to investigate the separation characteristics under typical RP gradient conditions. Column performance was compared under standard (identical) and optimized RP chromatographic conditions. The gradient compositions utilized with the C-CP fiber columns are similar to those used with conventional columns, employing flow rates in the 1-6 mL/min range and gradient rates of approximately 1%/min. The packed-bed column was operated as prescribed by the column manufacturer. The retention factor (k'), separation factor (alpha), resolution (Rs), asymmetry factor (As), elution order, and peak capacity values of a four protein separations performed on the C-CP fiber columns are compared to the same separation on the C4 column. One unique feature observed here is the lessening of the percentage of organic modifier necessary to elute the proteins from the fiber phases with increased linear velocity. The potential contribution of the different stationary phases to protein denaturation was evaluated through a spectrophotometric enzymatic activity assay. The repeatability of retention times under both sets of conditions for six consecutive injections of lysozyme on each C-CP fiber column is < or =1.5% RSD. The column-to-column reproducibility of retention times for three columns of each fiber type is also < or =1.5% RSD. The overall performance of the C-CP fiber columns was comparable to the conventional column used in these studies. Basic characteristics demonstrated here suggested further developments in the areas of ultrafast protein

  12. Use of a Novel Sub-2 µm Silica Hydride Vancomycin Stationary Phase in Nano-Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers.

    PubMed

    Rocchi, Silvia; Fanali, Chiara; Fanali, Salvatore

    2015-11-01

    A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano-liquid chromatography (nano-LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns-AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74-4.17 and 1.39-1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41-6.09 and α 1.28-2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. © 2015 Wiley Periodicals, Inc.

  13. Exploring chiral separation of 3-carboxamido-5-aryl isoxazole derivatives by supercritical fluid chromatography on amylose and cellulose tris dimethyl- and chloromethyl phenylcarbamate polysaccharide based stationary phases.

    PubMed

    Zehani, Yasmine; Lemaire, Lucas; Ghinet, Alina; Millet, Régis; Chavatte, Philippe; Vaccher, Claude; Lipka, Emmanuelle

    2016-10-07

    Four polysaccharide based chiral stationary phases were chosen, two chlorinated: Lux™ Amylose-2 (tris-5-chloro-2-methylphenylcarbamate of amylose) and Lux™ Cellulose-2 (tris-3-chloro-4-methylphenylcarbamate of cellulose) and two methylated: Chiralpak(®) AD-H (tris-3,5-dimethylphenylcarbamate of amylose) and Chiralcel(®) OD-H (tris-3,5-dimethylphenylcarbamate of cellulose) to separate four 3-carboxamido-5-aryl isoxazole derivatives by supercritical fluid chromatography. The effect of chiral stationary phase, co-solvent nature (MeOH, EtOH, 2-PrOH and ACN) and percentage (10-20%), temperature (20-45°C) and chemical structure of the compounds on retention, resolution and elution order were thoroughly studied. In addition, thermodynamic parameters were determined from the linear portion of the Van't Hoff plots. For all the derivatives, the Lux™ Cellulose-2 and Chiralpak(®) AD-H provided excellent resolutions (Rs=9.78) in short run time (under 6min). The preparation of about 10mg of each of the eight enantiomers was achieved successfully on a Chiralpak(®) AD-H with various percentages of ethanol as a co-solvent. Lastly, the enantiomeric purity of each of the eight individual enantiomer generated was determined and found higher than 98%.

  14. The use of Stationary Phase Optimized Selectivity Liquid Chromatography for the development of herbal fingerprints to detect targeted plants in plant food supplements.

    PubMed

    Deconinck, E; Djiogo, C A Sokeng; Kamugisha, A; Courselle, P

    2017-08-01

    The consumption of plant food supplements is increasing steadily and more and more, these products are bought through internet. Often the products sold through internet are not registered or declared with a national authority, meaning that no or minimal quality control is performed and that they could contain herbs or plants that are regulated. Stationary Phase Optimized Selectivity Liquid Chromatography (SOS-LC) was evaluated for the development of specific fingerprints, to be used for the detection of targeted plants in plant food supplements. Three commonly used plants in plant food supplements and two regulated plants were used to develop fingerprints with SOS-LC. It was shown that for all plants specific fingerprints could be obtained, allowing the detection of these targeted plants in triturations with different herbal matrices as well as in real samples of suspicious supplements seized by the authorities. For three of the five plants a more specific fingerprint was obtained, compared to the ones developed on traditional columns described in literature. It could therefore be concluded that the combination of segments of different types of stationary phases, as used in SOS-LC, has the potential of becoming a valuable tool in the quality control and the identification of crude herbal or plant material and in the detection of regulated plants in plant food supplements or other herbal preparations. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Analysis of N'-nitrosonornicotine enantiomers in human urine by chiral stationary phase liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry.

    PubMed

    Yang, Jing; Carmella, Steven G; Hecht, Stephen S

    2017-02-15

    We have developed a chiral stationary phase liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method to investigate the enantiomeric composition of low parts per trillion amounts of the carcinogen N'-nitrosonornicotine (NNN) in the urine of cigarette smokers and smokeless tobacco users. (S)-NNN is the major enantiomer in tobacco and is more carcinogenic than (R)-NNN in rats, but no data are available on the enantiomeric composition of NNN in humans. The method used [(13)C6]NNN as an internal standard and [pyridine-D4]nornicotine to monitor possible artifactual formation of NNN, which was found to be less than 2% of the quantified NNN. The enantiomeric composition of NNN (20.5±27.1fmol/mL urine) in 20 cigarette smokers was 67±5% (S)-NNN while that in 10 smokeless tobacco users (67.1±56.7 fmol/mL urine) was 56±3% (S)-NNN. These results demonstrate that the highly carcinogenic (S)-NNN is the major enantiomer in human urine, and that the enantiomeric composition of NNN in human urine is remarkably similar to that in cigarette smoke and smokeless tobacco. This is the first study to combine chiral stationary phase separations with nanoelectrospray ionization and high resolution tandem mass spectrometry to quantify trace levels of enantiomeric metabolites in human urine.

  16. Evaluation and comparison of n-alkyl chain and polar ligand bonded stationary phases for protein separation in reversed-phase liquid chromatography.

    PubMed

    Ding, Ling; Guo, Zhimou; Xiao, Yuansheng; Xue, Xingya; Zhang, Xiuli; Liang, Xinmiao

    2014-09-01

    Protein retention is very sensitive to the change of solvent composition in reversed-phase liquid chromatography for so called "on-off" mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3-chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3-chloropropyl trichlorosilane ligand bonded column and n-alkyl chain modified (C2, C4, C8) stationary phases were elucidated by the retention equation l nk=a+cC(B). Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3-chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3-chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3-chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the "on-off" problem and optimization in protein separation.

  17. Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.

    PubMed

    Valeja, Santosh G; Xiu, Lichen; Gregorich, Zachery R; Guner, Huseyin; Jin, Song; Ge, Ying

    2015-01-01

    To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.

  18. Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

    PubMed

    Periat, Aurélie; Kohler, Isabelle; Thomas, Aurélien; Nicoli, Raul; Boccard, Julien; Veuthey, Jean-Luc; Schappler, Julie; Guillarme, Davy

    2016-03-25

    Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples

  19. Investigation on the preparation and chromatographic behavior of a new para-tert-butylcalix[4]arene-1,2-crown-4 stationary phase for high performance liquid chromatography.

    PubMed

    Hu, Kai; Zhao, Wenjie; Wen, Fuyong; Liu, Junwei; Zhao, Xiaolan; Xu, Zhanhui; Niu, Bailin; Ye, Baoxian; Wu, Yangjie; Zhang, Shusheng

    2011-07-15

    In the present work, a new para-tert-butylcalix[4]arene-1,2-crown-4 bonded silica stationary phase (CBS4-4) was synthesized, structurally characterized, and employed to separate polycyclic aromatic hydrocarbons (PAHs), phenols, aromatic amines, benzoic acid and its derivatives. The chromatographic behaviors of the prepared stationary phase were investigated and compared with ODS. The effects of methanol concentrations on the retention index show that CBS4-4 exhibits high selectivity for the above analytes. The separation mechanisms based on the different interactions between calixarene and the analytes were discussed. With the assistance of quantum chemistry calculation, the interaction Gibbs free energy change ΔG(solv) (in the mobile phase) of p, m and o-phenylenediamine positional isomers and para-tert-butylcalix[4]arene-1,2-crown-4 were obtained. The ΔG(solv) values were consistent with the retention behavior of p, m and o-phenylenediamine on the CBS4-4. According to the chromatographic data, it can be concluded that the selectivity of CBS4-4 for analytes is mainly ascribed to hydrophobic interaction, accompanied by other effects such as hydrogen bonding interaction, π-π and inclusion interaction. The CBS4-4 column has been successfully employed for the analysis of benzoic acid in Sprite drink. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Separations of substituted benzenes and polycyclic aromatic hydrocarbons using normal- and reverse-phase high performance liquid chromatography with UiO-66 as the stationary phase.

    PubMed

    Zhao, Wei-Wei; Zhang, Chao-Yan; Yan, Zeng-Guang; Bai, Li-Ping; Wang, Xiayan; Huang, Hongliang; Zhou, You-Ya; Xie, Yabo; Li, Fa-Sheng; Li, Jian-Rong

    2014-11-28

    Metal-organic frameworks (MOFs) have great potential for applications in chromatography due to their highly tailorable porous structures and unique properties. In this work, the stable MOF UiO-66 was evaluated as both a normal-phase (NP-) and a reverse-phase (RP-) stationary phase in the high performance liquid chromatography (HPLC) to separate substituted benzenes (SBs) and polycyclic aromatic hydrocarbons (PAHs). It was found that the mobile phase composition has a significant effect on the HPLC separation. Baseline RP-HPLC separations of xylene isomers; naphthalene and anthracene; naphthalene and chrysene; and naphthalene, fluorene, and chrysene were achieved using MeOH/H2O ratios of 80:20, 75:25, 85:15, and 75:25, respectively, on the UiO-66 column. Similarly, baseline NP-HPLC separations of xylene isomers and ethylbenzene; ethylbenzene, styrene, o-xylene, and m-xylene; and several PAHs were also obtained on the UiO-66 column with different mobile phase compositions. The relative standard deviations (RSDs) of retention time, peak height, peak area, and half peak width for five replicate separations of the tested analytes were within the ranges 0.2-0.4%, 0.2-1.6%, 0.7-3.9%, 0.4-1.1%, respectively. We also evaluated other critical HPLC parameters, including injected sample mass, column temperature, and the thermodynamic characters of both the RP-HPLC and the NP-HPLC separation processes. It was confirmed that the separation of SBs on a UiO-66 column was an exothermic process, controlled by both enthalpy change (ΔH) and entropy change (ΔS). The reverse shape selectivity, size selectivity, stacking effect, and electrostatic force played vital roles in the separations of these analytes. To the best of our knowledge, this method is one of the very few examples of using MOFs as the stationary phase in both NP-HPLC and RP-HPLC. MOF-based stationary phases may thus be applied in the separations and analyses of SBs and PAHs in environmental samples.

  1. Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces.

    PubMed

    Gospodarek, Adrian M; Sun, Weitong; O'Connell, John P; Fernandez, Erik J

    2014-12-05

    In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized.

  2. Separation of carbohydrates using hydrophilic interaction liquid chromatography.

    PubMed

    Fu, Qing; Liang, Tu; Li, Zhenyu; Xu, Xiaoyong; Ke, Yanxiong; Jin, Yu; Liang, Xinmiao

    2013-09-20

    A strategy was developed to rapidly evaluate chromatographic properties of hydrophilic interaction chromatography (HILIC) columns for separating carbohydrates. Seven HILIC columns (Silica, Diol, TSK Amide-80, XAmide, Click Maltose, Click β-CD, and Click TE-Cys columns) were evaluated by using three monosaccharide and seven disaccharides as probes. The influence of column temperature on the peak shape and tautomerization of carbohydrates, as well as column selectivity were investigated. The influence of surface charge property on the retention was also studied by using glucose, glucuronic acid, and glucosamine, which indicated that buffer salt concentration and pH value in mobile phase was necessary to control the ionic interactions between ionic carbohydrates and HILIC columns. According to evaluation results, the XAmide column was selected as an example to establish experimental schemes for separation of complex mixtures of oligosaccharide.

  3. Synthesis of cis-C-Iodo-N-Tosyl-Aziridines using Diiodomethyllithium: Reaction Optimization, Product Scope and Stability, and a Protocol for Selection of Stationary Phase for Chromatography

    PubMed Central

    2013-01-01

    The preparation of C-iodo-N-Ts-aziridines with excellent cis-diastereoselectivity has been achieved in high yields by the addition of diiodomethyllithium to N-tosylimines and N-tosylimine–HSO2Tol adducts. This addition-cyclization protocol successfully provided a wide range of cis-iodoaziridines, including the first examples of alkyl-substituted iodoaziridines, with the reaction tolerating both aryl imines and alkyl imines. An ortho-chlorophenyl imine afforded a β-amino gem-diiodide under the optimized reaction conditions due to a postulated coordinated intermediate preventing cyclization. An effective protocol to assess the stability of the sensitive iodoaziridine functional group to chromatography was also developed. As a result of the judicious choice of stationary phase, the iodoaziridines could be purified by column chromatography; the use of deactivated basic alumina (activity IV) afforded high yield and purity. Rearrangements of electron-rich aryl-iodoaziridines have been promoted, selectively affording either novel α-iodo-N-Ts-imines or α-iodo-aldehydes in high yield. PMID:23738857

  4. Spherical clay conglomerates:  a novel stationary phase for solid-phase extraction and "reversed-phase" liquid chromatography.

    PubMed

    Bucheli, T D; Müller, S R; Reichmuth, P; Haderlein, S B; Schwarzenbach, R P

    1999-06-01

    A new solid phase is presented to be used for the solid-phase extraction (SPE) of organic compounds from aqueous solutions and as a stationary phase for the separation of organic compounds in "reversed-phase" HPLC. The material consists of spherical clay conglomerates (SCCs) in the size ranges of 2-5, 5-10, and 10-20 μm. SCCs are especially well suited for the extraction and separation of aromatic compounds with electron-withdrawing substituents, because of the formation of specific electron donor-acceptor (EDA) complexes of such compounds with natural clay minerals. A series of nitroaromatic compounds (NACs), e.g., nitrophenols, and nitrotoluenes, served as probe substances for the characterization of the SPE with SCCs online coupled to a C18-HPLC-DAD system. Breakthrough volumes were > 1 L and method detection limits (MDLs) < 100 ng/L for compounds with moderate to high affinity towards clay minerals. The performance of the material is hardly affected by matrix effects and because of its excellent physical properties, i.e., regenerability and pressure-resistance, it meets the requirements for fully automated routine trace analysis of several primary pollutants, such as 6-methyl-2,4-dinitrophenol (DNOC) or 2,4,6-trinitrotoluene (TNT), in various natural waters. Offline SPE with SCCs was superior or equivalent to commercial SPE products for analysis of such compounds. Finally, SCCs are shown to be well suited as a stationary phase in reversed-phase HPLC. This opens a wide range of applications, e.g., as an easy and fast separation technique that is orthogonal to C18 reversed-phase HPLC.

  5. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    PubMed

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1 month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possessed a larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions.

  6. Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics.

    PubMed

    Kahsay, Getu; Song, Huiying; Van Schepdael, Ann; Cabooter, Deirdre; Adams, Erwin

    2014-01-01

    This paper presents a general overview of the application of hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics in different sample matrices including pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin, plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams, tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative LC mode for separating small polar compounds. Polar samples usually show good solubility in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex systems that elute near the void in reversed-phase chromatography. Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS hence its increased popularity in recent years. In this review, the retention mechanism in HILIC is briefly discussed and a list of important applications is provided including main experimental conditions and a brief summary of the results. The references provide a comprehensive overview and insight into the application of HILIC in antibiotics analysis.

  7. Application of Homochiral Alkylated Organic Cages as Chiral Stationary Phases for Molecular Separations by Capillary Gas Chromatography.

    PubMed

    Xie, Shengming; Zhang, Junhui; Fu, Nan; Wang, Bangjin; Hu, Cong; Yuan, Liming

    2016-11-08

    Molecular organic cage compounds have attracted considerable attention due to their potential applications in gas storage, catalysis, chemical sensing, molecular separations, etc. In this study, a homochiral pentyl cage compound was synthesized from a condensation reaction of (S,S)-1,2-pentyl-1,2-diaminoethane and 1,3,5-triformylbenzene. The imine-linked pentyl cage diluted with a polysiloxane (OV-1701) was explored as a novel stationary phase for high-resolution gas chromatographic separation of organic compounds. Some positional isomers were baseline separated on the pentyl cage-coated capillary column. In particular, various types of enantiomers including chiral alcohols, esters, ethers and epoxides can be resolved without derivatization on the pentyl cage-coated capillary column. The reproducibility of the pentyl cage-coated capillary column for separation was investigated using nitrochlorobenzene and styrene oxide as analytes. The results indicate that the column has good stability and separation reproducibility after being repeatedly used. This work demonstrates that molecular organic cage compounds could become a novel class of chiral separation media in the near future.

  8. Dynamic evaluation of polypropylene capillary-channeled fibers as a stationary phase in high-performance liquid chromatography.

    PubMed

    Randunu, K Manoj; Dimartino, Simone; Marcus, R Kenneth

    2012-12-01

    Polypropylene (PP) capillary-channeled polymer (C-CP) fiber stationary phases are investigated for applications in HPLC. Specifically, the roles that fiber size and shape, linear velocity, interstitial fraction, and column inner diameter play in separation efficiency were evaluated using a uracil and butylparaben mixture eluted under isocratic conditions. Four fiber types, having nominal diameters ranging from 30 to 65 μm, were used in 250 mm × 2.1 mm columns. Optimum flow characteristics, as judged by plate height and resolution, were observed for 40 μm diameter PP C-CP fibers packed at an interstitial fraction of ~0.63, over a broad range of linear velocities (~2 to 37 mm/s). The influence of column inner diameter was studied on 1.5, 2.1, and 4.6 mm columns packed at the optimal interstitial fraction. The best performing column in terms of plate height and resolution was the 2.1 mm inner diameter. C-CP columns were also evaluated for the separation of a protein mixture composed of ribonuclease A, cytochrome c, and transferrin. Results obtained with the biomacromolecules mixture validate the optimal structural and operative conditions determined with the small solutes, laying the groundwork towards biomacromolecule applications, focusing more on the chemical aspects of separations. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Enantiomeric separations of α-aryl ketones with cyclofructan chiral stationary phases via high performance liquid chromatography and supercritical fluid chromatography.

    PubMed

    Breitbach, Anthony S; Lim, Yeeun; Xu, Qing-Long; Kürti, László; Armstrong, Daniel W; Breitbach, Zachary S

    2016-01-04

    Normal phase chiral HPLC and SFC methods are presented for the enantiomeric separation of 21 α-aryl ketones with a unique class of chiral stationary phases (CSPs) based on cyclofructans (CFs). Separations were achieved for all but 2 analytes, with 17 compounds attaining baseline separation having resolution values up to 4.0. Most separations obtained in HPLC could be transferred to SFC, but the HPLC resolutions were generally better due to greater enantiomeric selectivity values. A structure-separation relationship (SSR) was developed to identify important structural features for separation of this class of compounds using CF-based CSPs. Preliminary studies are also presented that demonstrate the utility of the CF CSPs to investigate the base-induced enantiomerization of α-aryl ketones. It was demonstrated that even small amounts of base (0.01%v/v) in the mobile phase results in rapid, on-column, enantiomerization. Lastly, CSPs composed of superficially porous particles were used to achieve comparable separations of this class of chiral compounds, but at a fraction of the analysis time compared to CSPs composed of fully porous particles. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Overdamped motion of interacting particles in general confining potentials: time-dependent and stationary-state analyses

    NASA Astrophysics Data System (ADS)

    Ribeiro, M. S.; Nobre, F. D.; Curado, E. M. F.

    2012-12-01

    By comparing numerical and analytical results, it is shown that a system of interacting particles under overdamped motion is very well described by a nonlinear Fokker-Planck equation, which can be associated with nonextensive statistical mechanics. The particle-particle interactions considered are repulsive, motivated by three different physical situations: (i) modified Bessel function, commonly used in vortex-vortex interactions, relevant for the flux-front penetration in disordered type-II superconductors; (ii) Yukawa-like forces, useful for charged particles in plasma, or colloidal suspensions; (iii) derived from a Gaussian potential, common in complex fluids, like polymer chains dispersed in a solvent. Moreover, the system is subjected to a general confining potential, φ( x) = ( α| x| z )/ z ( α > 0 , z > 1), so that a stationary state is reached after a sufficiently long time. Recent numerical and analytical investigations, considering interactions of type (i) and a harmonic confining potential ( z = 2), have shown strong evidence that a q-Gaussian distribution, P( x,t), with q = 0, describes appropriately the particle positions during their time evolution, as well as in their stationary state. Herein we reinforce further the connection with nonextensive statistical mechanics, by presenting numerical evidence showing that: (a) in the case z = 2, different particle-particle interactions only modify the diffusion parameter D of the nonlinear Fokker-Planck equation; (b) for z ≠ 2, all cases investigated fit well the analytical stationary solution P st( x), given in terms of a q-exponential (with the same index q = 0) of the general external potential φ( x). In this later case, we propose an approximate time-dependent P( x,t) (not known analytically for z ≠ 2), which is in very good agreement with the simulations for a large range of times, including the approach to the stationary state. The present work suggests that a wide variety of physical phenomena

  11. Two-dimensional hydrophilic interaction chromatography × reversed-phase liquid chromatography for the preparative isolation of potential anti-hepatitis phenylpropanoids from Salvia prattii.

    PubMed

    Dang, Jun; Shao, Yun; Zhao, Jianqiang; Mei, Lijuan; Tao, Yanduo; Wang, Qilan; Zhang, Li

    2016-09-01

    Traditional Tibetan medicine is important for discovery of drug precursors. However, knowledge of the chemical composition of traditional Tibetan medicines is very limited due to the lack of appropriate chromatographic purification methods. In the present work, Salvia prattii was taken as an example, and an off-line hydrophilic interaction liquid chromatography/reversed-phase liquid chromatography preparative method was developed for the purification of phenylpropanoids with high purity from a crude sample of Salvia prattii. Based on the separation results of four different chromatographic stationary phases, the first-dimensional preparation was performed on an XAmide preparative column with the crude sample concentration of 62.0 mg/mL, and five main fractions were obtained from the 12.4 g crude sample with a recovery of 54.8%. An XCharge C18 preparative column was applied in the second-dimensional preparation to further isolate the phenylpropanoids from the redissolved first-dimensional fractions with concentration of approximately 50.0 mg/mL. The purities of the phenylpropanoids isolated from the crude sample of Salvia prattii were higher than 98%, indicating that the method was efficient for the purification of phenylpropanoids with high purity from Salvia prattii. Additionally, this method showed great potential in the preparation of phenylpropanoids and can serve as a good example for the purification of phenylpropanoids from other plant materials. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Fabrication of ZIF-8@SiO2 core-shell microspheres as the stationary phase for high-performance liquid chromatography.

    PubMed

    Fu, Yan-Yan; Yang, Cheng-Xiong; Yan, Xiu-Ping

    2013-09-27

    The unique features of high porosity, shape selectivity, and multiple active sites make metal-organic frameworks (MOFs) promising as novel stationary phases for high-performance liquid chromatography (HPLC). However, the wide particle size distribution and irregular shape of conventional MOFs lead to lower column efficiency of such MOF-packed columns. Herein, the fabrication of monodisperse MOF@SiO2 core-shell microspheres as the stationary phase for HPLC to overcome the above-mentioned problems is reported. Zeolitic imidazolate framework 8 (ZIF-8) was used as an example of MOFs due to its permanent porosity, uniform pore size, and exceptional chemical stability. Unique carboxyl-modified silica spheres were used as the support to grow the ZIF-8 shell. The fabricated monodisperse ZIF-8@SiO2 packed columns (5 cm long × 4.6 mm i.d.) show high column efficiency (23,000 plates m(-1) for bisphenol A) for the HPLC separation of endocrine-disrupting chemicals (bisphenol A, β-estradiol, and p-(tert-octyl)phenol) and pesticides (thiamethoxam, hexaflumuron, chlorantraniliprole, and pymetrozine) within 7 min with good relative standard deviations for 11 replicate separations of the analytes (0.01-0.39, 0.65-1.7, 0.70-1.3, and 0.17-0.91% for retention time, peak area, peak height, and half peak width, respectively). The ZIF-8@SiO2 microspheres combine the advantages of the good column packing properties of the uniform monodisperse silica microspheres and the separation ability of the ZIF-8 crystals. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Comparison between polymerized ionic liquids synthesized using chain-growth and step-growth mechanisms used as stationary phase in gas chromatography.

    PubMed

    Roeleveld, Kevin; David, Frank; Lynen, Frédéric

    2016-06-17

    In this study the merits of polymerized imidazolium based ionic liquid (PIL) stationary phases obtained via condensation and free radical polymerizations are compared as stationary phases in gas chromatography (GC). Poly(1-vinyl-3-butyl-imidazolium - bis(trifluoromethane)sulfonamide) (poly(ViC4Im(+) NTf2(-))) was obtained via a chain-growth mechanism while poly(propylimidazolium-NTf2) (poly(C3Im(+) NTf2(-))) was synthesized via a step-growth polymerization. The thermal stability of both polymers was assessed using thermal gravimetric analysis and compared with bleeding profiles obtained from the statically coated GC columns (30m×0.25mm×0.25μm). The performance was compared to what could be obtained on commercially available 1,5-di(2,3-dimethylimidazolium)pentane(2+) 2NTf2(-) (SLB-IL111) ionic liquid based columns. It was observed that the step-growth polymer was more thermally stable, up to 325°C, while the chain-growth polymer showed initial degradation at 250°C. Both polymers allowed reaching minimal plate heights of 0.400-0.500mm for retained solutes such as benzaldehyde, acetophenone, 1-methylnaphthalene and aniline. Assessment of the McReynolds constants illustrated that the polarity of the step-growth polymer was similar to the SLB-IL111 column, while displaying improved column stability. The PIL phases and particularly the so far little studied condensation based polymer shows particular retention and satisfactory column performance for polar moieties such as esters, amine and carbonyl functionalities.

  14. Chromatography

    MedlinePlus

    Chromatography is a way of separating two or more chemical compounds. Chemical compounds are chemicals that are ... of chemical compound. There are different kinds of chromatography. These include gas, high pressure liquid, or ion ...

  15. Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase.

    PubMed

    Dolzan, Maressa D; Spudeit, Daniel A; Breitbach, Zachary S; Barber, William E; Micke, Gustavo A; Armstrong, Daniel W

    2014-10-24

    A new HILIC stationary phase comprised of native cyclofructan-6 (CF6) bonded to superficially porous silica particles (2.7μm) was developed. Its performance was evaluated and compared to fully porous silica particles with 5μm (commercially available as FRULIC-N) and 3μm diameters. Faster and more efficient chromatography was achieved with the superficially porous particles (SPPs). The columns were also evaluated in the normal phase mode. The peak efficiency, analysis time, resolution, and overall separation capabilities in both HILIC and normal phase modes were compared. The analysis times using the superficially porous based column in HILIC mode were shorter and the theoretical plates/min were higher over the entire range of flow rates studied. The column containing the superficially porous particles demonstrated higher optimum flow rates than the fully porous particle packed columns. At higher flow rates, the advantages of the superficially porous particles was more pronounced in normal phase separations than in HILIC, clearly demonstrating the influence that the mode of chromatography has on band broadening. However, the minimum reduced plate heights (hmin) were typically lower in HILIC than in the normal phase mode. Overall, the superficially porous particle based CF6 column showed clear advantages over the fully porous particle columns, in terms of high throughput and efficient separations of polar compounds in the HILIC mode.

  16. Determination of inorganic pharmaceutical counterions using hydrophilic interaction chromatography coupled with a Corona CAD detector.

    PubMed

    Huang, Z; Richards, M A; Zha, Y; Francis, R; Lozano, R; Ruan, J

    2009-12-05

    A simple generic approach was investigated for the determination of inorganic pharmaceutical counterions in drug substances using conventional high performance liquid chromatographic (HPLC) instruments. An intuitive approach combined Corona charged aerosol detection (CAD) with a polymer-based zwitterionic stationary phase in the hydrophilic interaction chromatography (HILIC) mode. Two generic methods based on this HILIC/CAD technique were developed to quantitate counterions such as Cl-, Br-, SO(4)(2-), K+, Ca2+ and Mg2+ in different pharmaceutical compounds. The development and capability of this HILIC/CAD technique analysis were examined. HILIC/CAD was compared to ion chromatography (IC), the most commonly used methodology for pharmaceutical counterion analysis. HILIC/CAD was found to have significant advantages in terms of: (1) being able to quantitate both anions and cations simultaneously without a need to change column/eluent or detection mode; (2) imposing much less restriction on the allowable organic percentage of the eluents than IC, and therefore being more appropriate for analysis of counterions of poorly water-soluble drugs; (3) requiring minimal training of the operating analysts. The precision and accuracy of counterion analysis using HILIC/CAD was not compromised. A typical precision of <2.0% was observed for all tested inorganic counterions; the determinations were within 2.0% relative to the theoretical counterion amount in the drug substance. Additionally, better accuracy was shown for Cl- in several drug substances as compared to IC. The main drawback of HILIC/CAD is its unsuitability for many of the current silica-based HILIC columns, because slight dissolution of silica leads to high baseline noise in the CAD detector. As a result of the universal detection characteristics of Corona CAD and the unique separation capabilities of a zwitterionic stationary phase, an intuitive and robust HPLC method was developed for the generic determination of

  17. Cell membrane chromatography competitive binding analysis for characterization of α1A adrenoreceptor binding interactions.

    PubMed

    Du, Hui; Ren, Jing; Wang, Sicen; He, Langchong

    2011-07-01

    A new high α(1A) adrenoreceptor (α(1A)AR) expression cell membrane chromatography (CMC) method was developed for characterization of α(1A)AR binding interactions. HEK293 α(1A) cell line, which expresses stably high levels of α(1A)AR, was used to prepare the stationary phase in the CMC model. The HEK293 α(1A)/CMC-offline-HPLC system was applied to specifically recognize the ligands which interact with the α(1A)AR, and the dissociation equilibrium constants (K (D)) obtained from the model were (1.87 ± 0.13) × 10(-6) M for tamsulosin, (2.86 ± 0.20) × 10(-6) M for 5-methylurapidil, (3.01 ± 0.19) × 10(-6) M for doxazosin, (3.44 ± 0.19) × 10(-6) M for terazosin, (3.50 ± 0.21) × 10(-6) M for alfuzosin, and (7.57 ± 0.31) × 10(-6) M for phentolamine, respectively. The competitive binding study between tamsulosin and terazosin indicated that the two drugs interacted at the common binding site of α(1A)AR. However, that was not the case between tamsulosin and oxymetazoline. The results had a positive correlation with those from radioligand binding assay and indicated that the CMC method combined modified competitive binding could be a quick and efficient way for characterizing the drug-receptor interactions.

  18. Increasing selectivity in comprehensive three-dimensional gas chromatography via an ionic liquid stationary phase column in one dimension.

    PubMed

    Siegler, W Christopher; Crank, Jeffery A; Armstrong, Daniel W; Synovec, Robert E

    2010-04-30

    Recent advances in improving the selectivity and performance for a comprehensive, three-dimensional (3D) gas chromatograph (GC(3)) instrument are described. With GC(3), two six-port diaphragm valves are utilized as the interfaces between three, in-series capillary columns housed in a standard GC instrument fitted with a high data acquisition rate flame ionization detector (FID). Modulation periods for sampling from one column to the next are set so that sufficient slices (i.e., modulations) are acquired by the subsequent dimension resulting in comprehensive data. We present GC(3) instrumentation with significantly higher 3D peak capacity than previously reported. An average peak capacity production (i.e., per time) of 180 resolved peaks per minute was experimentally achieved for three representative analytes in a 3D diesel sample separation. This peak capacity production is about 4 times higher than our previous report. We also demonstrate the significant benefit of the added chemical selectivity of the three column GC(3) instrument relative to a two column GC x GC instrument, in which one of the three columns is a triflate ionic liquid stationary phase column with a high selectivity for phosphonated compounds (i.e., di-methyl-methyl phosphonate, di-ethyl-methyl phosphonate and di-isopropyl-methyl phosphonate). Using all three separation dimensions, the 2D separation fingerprint of a diesel sample is simultaneously obtained along with selective information regarding the phosphonated compounds in the diesel samples in the additional dimension. 2010. Published by Elsevier B.V.

  19. Aniline-modified porous graphitic carbon for hydrophilic interaction and attenuated reverse phase liquid chromatography.

    PubMed

    Iverson, Chad D; Lucy, Charles A

    2014-12-19

    Most stationary phases for hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC) are based on silica. Porous graphitic carbon (PGC) is an attractive alternative to silica-based phases due to its chemical and thermal stability, and unique selectivity. However, native PGC is strongly hydrophobic and in some instances excessively retentive. PGC particles with covalently attached aniline groups (Dimethylaniline-PGC and Aniline-PGC) were synthesized to alter the surface polarity of PGC. First, the diazonium salt of N,N-dimethyl-p-phenylenediamine or 4-nitroaniline was adsorbed onto the PGC surface. The adsorbed salt was reduced with sodium borohydride and (Aniline-PGC only) the nitro group was further reduced with iron powder to the aniline. X-ray photoelectron spectroscopy confirmed the surface functionalities and that these moieties were introduced to the surface at concentrations of 0.9 and 2.1molecules/nm(2), respectively. These modified PGC phases (especially Aniline-PGC) were evaluated as HILIC and reversed phases. The Dimethylaniline-PGC phase displayed only weak HILIC retention of phenolic solutes. In contrast, the Aniline-PGC phase displayed up to nearly a 7-fold increase in HILIC retention vs. an aniline-silica phase and selectivity that differed from 10 other HILIC phases. Introduction of aniline groups to the PGC surface reduced the RPLC retentivity of PGC up to more than 5-fold and improved the separation efficiency up to 6-fold. The chromatographic performance of Aniline-PGC is demonstrated by separations of nucleotides, nucleosides, carboxylic acids, basic pharmaceuticals, and other compounds.

  20. Ionic liquid-based zwitterionic organic polymer monolithic column for capillary hydrophilic interaction chromatography.

    PubMed

    Wang, Tingting; Chen, Yihui; Ma, Junfeng; Zhang, Xiaodan; Zhang, Lihua; Zhang, Yukui

    2015-08-21

    In the current study, a novel ionic liquid-based zwitterionic organic polymer monolithic column was developed by copolymerizing 1-vinyl-3-(butyl-4-sulfonate) imidazolium, acrylamide and N,N'-methylenebisacrylamide in a quaternary porogenic solvent consisting of formamide, dimethyl sulphoxide, polyethylene glycol 8000 and polyethylene glycol 10,000 for capillary hydrophilic interaction chromatography. The monolithic stationary phase was optimized by adjusting the amount of monomer in the polymerization solution along with the composition of porogenic solvent. The optimized monolith exhibited excellent selectivity and favorable retention for nucleosides and benzoic acid derivatives. The primary factors affecting the separation efficiency of the monolithic column (including acetonitrile content, pH, and buffer salt concentration in the mobile phase) have been thoroughly evaluated. Excellent reproducibility of the retention times for five nucleosides was achieved, with relative standard deviations of run-to-run (n = 3), column-to-column (n = 3) and batch-to-batch (n = 3) in the range of 0.18-0.48%, 2.33-4.20% and 3.07-6.50%, respectively.

  1. Comparison of peak shape in hydrophilic interaction chromatography using acidic salt buffers and simple acid solutions.

    PubMed

    Heaton, James C; Russell, Joseph J; Underwood, Tim; Boughtflower, Robert; McCalley, David V

    2014-06-20

    The retention and peak shape of neutral, basic and acidic solutes was studied on hydrophilic interaction chromatography (HILIC) stationary phases that showed both strong and weak ionic retention characteristics, using aqueous-acetonitrile mobile phases containing either formic acid (FA), ammonium formate (AF) or phosphoric acid (PA). The effect of organic solvent concentration on the results was also studied. Peak shape was good for neutrals under most mobile phase conditions. However, peak shapes for ionised solutes, particularly for basic compounds, were considerably worse in FA than AF. Even neutral compounds showed deterioration in performance with FA when the mobile phase water concentration was reduced. The poor performance in FA cannot be entirely attributed to the negative impact of ionic retention on ionised silanols on the underlying silica base materials, as results using PA at lower pH (where their ionisation is suppressed) were inferior to those in AF. Besides the moderating influence of the salt cation on ionic retention, it is likely that salt buffers improve peak shape due to the increased ionic strength of the mobile phase and its impact on the formation of the water layer on the column surface. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry.

    PubMed

    Ito, Jun; Herter, Thomas; Baidoo, Edward E K; Lao, Jeemeng; Vega-Sánchez, Miguel E; Michelle Smith-Moritz, A; Adams, Paul D; Keasling, Jay D; Usadel, Björn; Petzold, Christopher J; Heazlewood, Joshua L

    2014-03-01

    Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is limited by difficulties in performing sensitive quantification of many involved compounds. Hydrophilic interaction liquid chromatography is a useful technique for the analysis of hydrophilic metabolites from complex biological extracts and forms the basis of this method to quantify plant cell wall precursors. A zwitterionic silica-based stationary phase has been used to separate hydrophilic nucleotide sugars involved in cell wall biosynthesis from milligram amounts of leaf tissue. A tandem mass spectrometry operating in selected reaction monitoring mode was used to quantify nucleotide sugars. This method was highly repeatable and quantified 12 nucleotide sugars at low femtomole quantities, with linear responses up to four orders of magnitude to several 100pmol. The method was also successfully applied to the analysis of purified leaf extracts from two model plant species with variations in their cell wall sugar compositions and indicated significant differences in the levels of 6 out of 12 nucleotide sugars. The plant nucleotide sugar extraction procedure was demonstrated to have good recovery rates with minimal matrix effects. The approach results in a significant improvement in sensitivity when applied to plant samples over currently employed techniques. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Isolation of soybean protein P34 from oil bodies using hydrophobic interaction chromatography

    PubMed Central

    Sewekow, Eva; Keßler, Lars Christian; Seidel-Morgenstern, Andreas; Rothkötter, Hermann-Josef

    2008-01-01

    Background Soybeans play a prominent role in allergologic research due to the high incidence of allergic reactions. For detailed studies on specific proteins it is necessary to have access to a large amount of pure substance. Results In this contribution, a method for purifying soybean (Glycine max) protein P34 (also called Gly m Bd 30 K or Gly m 1) using hydrophobic interaction chromatography is presented. After screening experiments using 1 mL HiTrap columns, Butyl Sepharose 4 FF was selected for further systematic investigations. With this stationary phase, suitable operation conditions for two-step gradient elution using ammonium sulphate were determined experimentally. The separation conditions obtained in a small column could be scaled up successfully to column volumes of 7.5 and 75 mL, allowing for high product purities of almost 100% with a yield of 27% for the chromatographic separation step. Conditions could be simplified further using a onestep gradient, which gave comparable purification in a shorter process time. The identity of the purified protein was verified using in-gel digestion and mass spectrometry as well as immunological techniques. Conclusion With the technique presented it is possible to produce, within a short timeframe, pure P34, suitable for further studies where an example antigen is needed. PMID:18334018

  4. Mixed-mode chromatography and its applications to biopolymers.

    PubMed

    Yang, Yun; Geng, Xindu

    2011-12-09

    Mixed-mode chromatography is a type of chromatography in which a chromatographic stationary phase interacts with solutes through more than one interaction mode. This technique has been growing rapidly because of its advantages over conventional chromatography, such as its high resolution, high selectivity, high sample loading, high speed, and the ability to replace two conventionally corresponding columns in certain circumstances. In this work, some aspects of the development of mixed-mode chromatography are reviewed, such as stationary phase preparation, combinations of various separation modes, separation mechanisms, typical applications to biopolymers and peptides, and future prospects. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Chromatographic performance of synthetic polycrystalline diamond as a stationary phase in normal phase high performance liquid chromatography.

    PubMed

    Peristyy, Anton; Paull, Brett; Nesterenko, Pavel N

    2015-04-24

    The chromatographic properties of high pressure high temperature synthesised diamond (HPHT) are investigated in normal phase mode of high performance liquid chromatography. Purified nonporous irregular shape particles of average particles size 1.2 μm and specific surface area 5.1 m(2) g(-1) were used for packing 100×4.6 mm ID or 50×4.6 mm ID stainless steel columns. The retention behaviour of several classes of compounds including alkyl benzenes, polyaromatic hydrocarbons (PAH), alkylphenylketones, phenols, aromatic acids and bases were studied using n-hexane-2-propanol mixtures as mobile phase. The results are compared with those observed for microdispersed sintered detonation nanodiamond (MSDN) and porous graphitic carbon (PGC). HPHT diamond revealed distinctive separation selectivity, which is orthogonal to that observed for porous graphitic carbon; while selectivities of HPHT diamond and microdispersed sintered detonation nanodiamonds are similar. Owing to non-porous particle nature, columns packed with high pressure high temperature diamond exhibited excellent mass transfer and produce separations with maximum column efficiency of 128,200 theoretical plates per meter.

  6. Determination of pore size distributions in capillary-channeled polymer fiber stationary phases by inverse size-exclusion chromatography and implications for fast protein separations.

    PubMed

    Wang, Zhengxin; Marcus, R Kenneth

    2014-07-18

    Capillary-channeled polymer (C-CP) fibers have been utilized as liquid chromatography stationary phases, primarily for biomacromolecule separations on the analytical and preparative scales. The collinear packing of the eight-channeled C-CP fibers provides for very efficient flow, allowing operation at high linear velocity (u>100mm s(-1)) and low backpressure (<2000psi) in analytical-scale separations. To take advantage of these fluid transport properties, there must not be mass transfer limitations as would be imposed by having an appreciably porous phase, wherein solute diffusion limits the overall mass transport rates. To better understand the physical nano-/micro- structure of C-CP fibers, inverse size exclusion chromatography (iSEC) has been employed to determine the pore size distribution (PSD) within C-CP fibers. A diversity of test species (from metal ions to large proteins) was used as probes under non-retaining conditions to obtain a response curve reflecting the apparent partition coefficient (Kd) versus hydrodynamic radii (rm). A mean pore radius (rp) of 4.2nm with standard deviation (sp) of ±1.1nm was calculated by fitting the Kd versus rm data to model equations with a Gaussian pore size distribution, and a pore radius of 4.0±0.1nm was calculated based on a log-normal distribution. The derived mean pore radius is much smaller than traditional support materials, with the standard deviation showing a relatively uniform pore distribution. van Deemter plots were analyzed to provide practical confirmation of the structural implications. Large molecules (e.g., proteins) that are fully excluded from pores have no significant C-terms in the van Deemter plots whereas small molecules that can access the pore volumes display appreciable C-terms, as expected. Fitting of retention data to the Knox equation suggests that the columns operate with a characteristic particle diameter (dp) of ∼53μm.

  7. Direct high-performance liquid chromatography enantioseparation of terazosin on an immobilised polysaccharide-based chiral stationary phase under polar organic and reversed-phase conditions.

    PubMed

    Ferretti, Rosella; Gallinella, Bruno; La Torre, Francesco; Zanitti, Leo; Turchetto, Luciana; Mosca, Antonina; Cirilli, Roberto

    2009-07-10

    High-performance liquid chromatography (HPLC) enantioseparation of terazosin (TER) was accomplished on the immobilised-type Chiralpak IC chiral stationary phase (CSP) under both polar organic and reversed-phase modes. A simple analytical method was validated using a mixture of methanol-water-DEA 95:5:0.1 (v/v/v) as a mobile phase. Under reversed-phase conditions good linearities were obtained over the concentration range 8.76-26.28 microg mL(-1) for both enantiomers. The limits of detection and quantification were 10 and 30 ng mL(-1), respectively. The intra- and inter-day assay precision was less than 1.66% (RSD%). The optimised conditions also allowed to resolve chiral and achiral impurities from the enantiomers of TER. The proposed HPLC method supports pharmacological studies on the biological effects of the both forms of TER and analytical investigations of potential drug formulations based on a single enantiomer. At the semipreparative scale, 5.3 mg of racemic sample were resolved with elution times less than 12 min using a mobile phase consisting of methanol-DEA 100:0.1 (v/v) and both enantiomers were isolated with a purity of > or = 99% enantiomeric excess (ee). The absolute configuration of TER enantiomers was assigned by comparison of the measured specific rotations with those reported in the literature.

  8. Enantiomeric separation of isochromene derivatives by high-performance liquid chromatography using cyclodextrin based stationary phases and principal component analysis of the separation data.

    PubMed

    Nanayakkara, Yasith S; Woods, Ross M; Breitbach, Zachary S; Handa, Sachin; Slaughter, LeGrande M; Armstrong, Daniel W

    2013-08-30

    Isochromene derivatives are very important precursors in the natural products industry. Hence the enantiomeric separations of chiral isochromenes are important in the pharmaceutical industry and for organic asymmetric synthesis. Here we report enantiomeric separations of 21 different chiral isochromene derivatives, which were synthesized using alkynylbenzaldehyde cyclization catalyzed by chiral gold(I) acyclic diaminocarbene complexes. All separations were achieved by high-performance liquid chromatography with cyclodextrin based (Cyclobond) chiral stationary phases. Retention data of 21 chiral compounds and 14 other previously separated isochromene derivatives were analyzed using principal component analysis. The effect of the structure of the substituents on the isochromene ring on enantiomeric resolution as well as the other separation properties was analyzed in detail. Using principal component analysis it can be shown that the structural features that contribute to increased retention are different from those that enhance enantiomeric resolution. In addition, principal component analysis is useful for eliminating redundant factors from consideration when analyzing the effect of various chromatographic parameters. It was found that the chiral recognition mechanism is different for the larger γ-cyclodextrin as compared to the smaller β-cyclodextrin derivatives. Finally this specific system of chiral analytes and cyclodextrin based chiral selectors provides an effective format to examine the application of principal component analysis to enantiomeric separations using basic retention data and structural features. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Enantioseparation of N-fluorenylmethoxycarbonyl alpha-amino acids on polysaccharide-derived chiral stationary phases by reverse mode liquid chromatography.

    PubMed

    Lee, Kyung-Ah; Yeo, Sujeong; Kim, Kyeong Ho; Lee, Wonjae; Kang, Jong Seong

    2008-04-14

    The enantioseparation of N-protected fluorenylmethoxycarbonyl (N-FMOC) alpha-amino acids was carried out on three polysaccharide-derived chiral stationary phases, such as cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD), amylose tris(3,5-dimethyl-phenylcarbamate) (Chiralpak AD) and cellulose tris(4-methylbenzoate) (Chiralcel OJ), and the influence of acetonitrile composition and pH of the eluents on the enantioseparation in reverse mode chromatography was examined. The best separation of the enantiomers was achieved with 40% acetonitrile in 50mM phosphate buffer at pH 2. However, increasing the composition of acetonitrile to 50% on Chiralcel OD yielded a considerable decrease of retention time with minimum loss of resolution. The elution order of N-FMOC alpha-amino acid enantiomers on Chiralcel OD and OJ were quite different, indicating that both phases could be used in a complementary manner for the separation of the enantiomers of N-FMOC alpha-amino acids. The positive relationship between the capacity factor of N-FMOC alpha-amino acids and the hydrophobicity of amino acids indicated that hydrophobicity plays an important role on the retention of the N-FMOC alpha-amino acids in the reverse mode.

  10. Thermally sensitive behavior explanation for unusual orthogonality observed in comprehensive two-dimensional gas chromatography comprising a single ionic liquid stationary phase.

    PubMed

    Nolvachai, Yada; Kulsing, Chadin; Marriott, Philip J

    2015-01-06

    In this study, a theoretical concept and method to achieve a degree of orthogonality in comprehensive two-dimensional gas chromatography (GC × GC) for separation of fatty acid methyl esters (FAME) by using a single ionic liquid (IL) stationary phase (1-phase-GC × GC) were established. The 1-phase system comprises a long IL column and shorter IL column of the same phase before and after the modulation region, operated under temperature-programmed conditions. Initial isothermal experiments employing six commercial IL columns were conducted at different temperatures. On the basis of the temperature-dependent linear solvation energy relationship (LSER) concept, SLB-IL111 exhibited the greatest thermal sensitivity and degree of difference over the tested temperature (T) range, so it was selected for investigation of the 1-phase-GC × GC mode. With the same temperature program, a significantly high degree of orthogonality was observed for the experiment, varied with column lengths. The switchable separation result, which inverts the retention of saturated and unsaturated FAME on the downstream column ((2)D), was achieved by varying column diameters and surface thicknesses of the IL-coated layers. These results were explained according to the corresponding LSER principles. Also, the time summation model was applied for the simulation of the observed 1-phase-GC × GC results.

  11. Enantiomers separation by nano-liquid chromatography: use of a novel sub-2 μm vancomycin silica hydride stationary phase.

    PubMed

    Rocchi, Silvia; Rocco, Anna; Pesek, Joseph J; Matyska, Maria T; Capitani, Donatella; Fanali, Salvatore

    2015-02-13

    A novel sub-2 μm chiral stationary phase (CSP) was prepared immobilizing vancomycin onto 1.8 μm diol hydride-based silica particles. The CSP was packed into fused silica capillaries of 75 μm i.d. with a length of 11 cm and evaluated by means of nano-liquid chromatography (nano-LC) using model compounds of both pharmaceutical and environmental interest (some non-steroidal anti-inflammatory drugs, β-blockers and herbicides). The study of the effect of the linear velocity of the mobile phase on chromatographic efficiency showed good enantioresolutions up to a value of 5.11 at the optimal linear velocity with efficiencies in terms of number of plates per meter in the range 51,650-68,330. The results were compared with the ones obtained employing 5 μm vancomycin modified diol-silica particles packed in capillaries of the same i.d. For the acidic analytes the sub-2 μm CSP showed better performances, the baseline chiral separation of several studied compounds occurred in an analysis time of less than 3 min. Column-to-column packing reproducibility (n=3) expressed as relative standard deviation was in the range 2.2-5.8% and 0.5-7.7% for retention times and peak areas, respectively.

  12. Application of a cholesterol stationary phase in the analysis of phosphorothioate oligonucleotides by means of ion pair chromatography coupled with tandem mass spectrometry.

    PubMed

    Studzińska, Sylwia; Krzemińska, Katarzyna; Szumski, Michał; Buszewski, Bogusław

    2016-07-01

    The main aim of this study was the investigation of the influence of several ion pair reagents towards both the retention and the mass spectrometry sensitivity of phosphorothioate oligonucleotides. A cholesterol stationary phase was applied for the first time in the analysis of this group of compounds. The mobile phase composition was modified by changing the concentration and the type of amines and acetates or 1,1,1,3,3,3-hexafluoroisopropanol. It has been shown that the increase of amines concentration results in the retention factor increase for each oligonucleotide, on each adsorbent. The only exception was the mobile phase composed of triethylamine and 1,1,1,3,3,3-hexafluoroisopropanol. This is a consequence of interactions taking place between a cholesterol molecule and an alcohol. This effect was convenient when the mass spectrometry detection was applied, since it allowed an increase in the sensitivity. Moreover, optimization of the mobile phase composition and its impact on the efficiency of ionization process and on the sensitivity in mass spectrometry were also presented. The optimization of this new method, based on cholesterol stationary phase coupled with mass spectrometry detection, was finally applied for the determination of phosphorothioate oligonucleotides impurity in a real sample. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Preparation and evaluation of a lysine-bonded silica monolith as polar stationary phase for hydrophilic interaction pressurized capillary electrochromatography.

    PubMed

    Huang, Guihua; Lian, Qiuyan; Zeng, Wencan; Xie, Zenghong

    2008-09-01

    A silica-based monolith as polar stationary phase was described for hydrophilic interaction pressurized capillary electrochromatography (HI-pCEC). The polar monolithic column was prepared by on-column reaction of lysine with epoxy groups on a gamma-glycidoxypropyltrimethosysilane-modified silica monolith. The stationary phase yielded strong hydrophilic interaction due to the slightly polar hydroxyl groups, and the strong polar lysine ligand with amino groups and carboxylic groups contained on the surface of the monolith. In order to evaluate the hydrophilic character of lysine ligand, the chromatographic behaviors of epoxy monolith (before lysine bonded) and diol monolith (hydroxyl groups contained) were also investigated. Two groups of comparative experiment were developed in terms of the separation of typical neutral non-polar and polar compounds performed in a mobile phase of aqueous-acetonitrile solution. Results showed that the lysine monolith was much more hydrophilic than the diol monolith, which presented less hydrophobic than the epoxy monolith. For further study on its hydrophilic character, the lysine monolith was demonstrated in the HI-pCEC mode for the separations of various polar compounds such as phenols, nucleic acid bases and nucleosides.

  14. On the interaction of stationary crossflow vortices and Tollmien-Schlichting waves in the boundary layer on a rotating disc

    NASA Technical Reports Server (NTRS)

    Bassom, Andrew P.; Hall, Philip

    1989-01-01

    There are many fluid flows where the onset of transition can be caused by different instability mechanisms which compete among themselves. The interaction is considered of two types of instability mode (at an asymptotically large Reynolds number) which can occur in the flow above a rotating disc. In particular, the interaction is examined between lower branch Tollmien-Schlichting (TS) waves and the upper branch, stationary, inviscid crossflow vortex whose asymptotic structure has been described by Hall (1986). This problem is studied in the context of investigating the effect of the vortex on the stability characteristics of a small TS wave. Essentially, it is found that the primary effect is felt through the modification to the mean flow induced by the presence of the vortex. Initially, the TS wave is taken to be linear in character and it is shown (for the cases of both a linear and a nonlinear stationary vortex) that the vortex can exhibit both stabilizing and destabilizing effects on the TS wave and the nature of this influence is wholly dependent upon the orientation of this latter instability. Further, the problem is examined with a larger TS wave, whose size is chosen so as to ensure that this mode is nonlinear in its own right. An amplitude equation for the evolution of the TS wave is derived which admits solutions corresponding to finite amplitude, stable, traveling waves.

  15. A new method for separation and determination of Cr(III) and Cr(VI) in water samples by high-performance liquid chromatography based on anion exchange stationary phase of ionic liquid modified silica.

    PubMed

    Sadeghi, Susan; Moghaddam, Ali Zeraatkar

    2015-12-01

    In this work, N-methylimidazolium-chloride ionic liquid functionalized silica was prepared and used as an anion-exchange stationary phase for separation of chromium species by high-performance liquid chromatography (HPLC) with UV detection at 200 nm. The Cr(VI) as HCr2O7(-) and chelated Cr(III) with potassium hydrogen phthalate (PHP) as Cr(PHP)2 (-) was retained on the prepared column and separated using a mobile phase composed of 5% methanol in 25 mM phosphate buffer at pH 6.5. Several variables affecting the chelation/separation steps were modeled by response surface methodology (RSM) using Box-Behnken (BBD) design. The significance of the independent variables and their interactions were tested by the analysis of variances (ANOVA) with 95% confidence limit. Under the optimized conditions, the Cr(III) and Cr(VI) anionic species were well separated with a single peak for each Cr species at retention times of 2.3 and 4.3 min, respectively. The relationship between the peak area and concentration was linear in the range of 0.025-30 for Cr(III) and 0.5-20 mg L(-1) for Cr(VI) with detection limits of 0.010 and 0.210 mg L(-1) for Cr(III) and Cr(VI), respectively. The proposed method was validated by simultaneous separation and determination of the Cr species in tap and underground water samples without impose to any pretreatment.

  16. Group separation of transplutonium and rare-earth elements by liquid chromatography with a free stationary phase using 2,4,6-Tris[ditolylphosphoryl]-1,3,5-triazine

    SciTech Connect

    Chmutova, M.K.; Ivanova, L.A.; Bodrin, G.B.

    1995-03-01

    Methods are developed for group separation of trace quantities of transplutonium (TPE) and weighable amounts of rare-earth elements (REE) by liquid chromatography with a free stationary phase in systems based on bifunctional neutral organophosphorus compounds. Using a stationary phase of 2,4,6-tris(ditolylphosphoryl)-1,3,5-triazine in CHCl{sub 3}, REE are first eluted by 0.5 M NH{sub 4}SCN-1 M HCl and then TPE by 0.025 M hydroxyethylidenediphosphonic acid in H{sub 2}O. The fractions contained {approximately} 100% of one of the groups without an impurity of the other. Use of the same eluents and a CHCl{sub 3} solution of tetraphenyl-methylenediphosphine dioxide as the stationary phase gave 95.4% pure REE and 97.5% pure TPE.

  17. Determining stationary-state quantum properties directly from system-environment interactions

    NASA Astrophysics Data System (ADS)

    Nicacio, F.; Paternostro, M.; Ferraro, A.

    2016-11-01

    Considering stationary states of continuous-variable systems undergoing an open dynamics, we unveil the connection between properties and symmetries of the latter and the dynamical parameters. In particular, we explore the relation between the Lyapunov equation for dynamical systems and the steady-state solutions of a time-independent Lindblad master equation for bosonic modes. Exploiting bona fide relations that characterize some genuine quantum properties (entanglement, classicality, and steerability), we obtain conditions on the dynamical parameters for which the system is driven to a steady state possessing such properties. We also develop a method to capture the symmetries of a steady state based on symmetries of the Lyapunov equation. All the results and examples can be useful for steady-state engineering processes.

  18. Insights into the retention mechanism of neutral organic compounds on polar chemically bonded stationary phases in reversed-phase liquid chromatography.

    PubMed

    Ali, Zahid; Poole, C F

    2004-10-15

    The solvation parameter model is used to characterize the retention properties of a 3-aminopropylsiloxane-bonded (Alltima amino), three 3-cyanopropylsiloxane-bonded (Ultrasphere CN, Ultremex-CN and Zorbax SB-CN), a spacer bonded propanediol (LiChrospher DIOL) and a multifunctional macrocyclic glycopeptide (Chirobiotic T) silica-based stationary phases with mobile phases containing 10 and 20% (v/v) methanol-water. The low retention on the polar chemically bonded stationary phases compared with alkylsiloxane-bonded silica stationary phases arises from the higher cohesion of the polar chemically bonded phases and an unfavorable phase ratio. The solvated polar chemically bonded stationary phases are considerably more hydrogen-bond acidic and dipolar/polarizable than solvated alkylsiloxane-bonded silica stationary phases. Selectivity differences are not as great among the polar chemically bonded stationary phases as they are between the polar chemically bonded phases and alkylsiloxane-bonded silica stationary phases.

  19. Chromatographic analysis of olopatadine in hydrophilic interaction liquid chromatography.

    PubMed

    Maksić, Jelena; Jovanović, Marko; Rakić, Tijana; Popović, Igor; Ivanović, Darko; Jančić-Stojanović, Biljana

    2015-01-01

    In this paper, chromatographic analysis of active substance olopatadine hydrochloride, which is used in eye drops as antihistaminic agent, and its impurity E isomer by hydrophilic interaction liquid chromatography (HILIC) and application of design of experiments (DoE) methodology are presented. In addition, benzalkonium chloride is very often used as a preservative in eye drops. Therefore, the evaluation of its chromatographic behavior in HILIC was carried out as well. In order to estimate chromatographic behavior and set optimal chromatographic conditions, DoE methodology was applied. After the selection of important chromatographic factors, Box-Behnken design was utilized, and on the basis of the obtained models factor effects were examined. Then, multi-objective robust optimization is performed aiming to obtain chromatographic conditions that comply with several quality criteria simultaneously: adequate and robust separation of critical peak pair and maximum retention of the first eluting peak. The optimal conditions are identified by using grid point search methodology. The experimental verification confirmed the adequacy of the defined optimal conditions. Finally, under optimal chromatographic conditions, the method was validated and applicability of the proposed method was confirmed. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography.

    PubMed

    Jiang, Zhengjin; Reilly, John; Everatt, Brian; Smith, Norman W

    2009-03-20

    A novel porous zwitterionic monolith was prepared by thermal co-polymerisation of 2-methacryloyloxyethyl phosphorylcholine (MPC) and ethylene glycol dimethacrylate (EDMA) within 100 microm I.D. capillaries. Mercury intrusion porosimetry, scanning electron microscopy (SEM), micro-HPLC (micro-HPLC), elemental analysis and zeta-potential analysis were used to evaluate the monolithic structure. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical hydrophilic liquid chromatography (HILIC) mechanism was observed at high organic solvent content (acetonitrile >60%). The phosphorylcholine (PC) functionality has both a positively charged quaternary ammonium and a negatively charged phosphate group. For charged analytes, a weak electrostatic interaction was also observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(MPC-co-EDMA) monolithic column. The optimised poly(MPC-co-EDMA) monolith showed very good selectivities for a range of polar test analytes, especially small peptides. This might be ascribed to the good biocompatibility of PC functionality. At low organic solvent content, baseline separation was also observed for a test mixture of seven alkylphenones by a reversed-phase separation mechanism.

  1. A comprehensive study to protein retention in hydrophobic interaction chromatography.

    PubMed

    Baca, Martyna; De Vos, Jelle; Bruylants, Gilles; Bartik, Kristin; Liu, Xiaodong; Cook, Ken; Eeltink, Sebastiaan

    2016-10-01

    The effect of different kosmotropic/chaotropic salt systems on retention characteristics of intact proteins has been examined in hydrophobic interaction chromatography (HIC). The performance was assessed using different column chemistries, i.e., polyalkylamide, alkylamine incorporating hydrophobic moieties, and a butyl chemistry. Selectivity in HIC is mainly governed by the salt concentration and by the molal surface tension increment of the salt. Typically, a linear relationship between the natural logarithm of the retention factor and the salt concentration is obtained. Using a 250mm long column packed with 5μm polyalkylamide functionalized silica particles and applying a 30min linear salt gradient, a peak capacity of 78 was achieved, allowing the baseline separation of seven intact proteins. The hydrophobicity index appeared to be a good indicator to predict the elution order of intact proteins in HIC mode. Furthermore, the effect of adding additives in the mobile phase, such as calcium chloride (stabilizing the 3D conformation of α-lactalbumin) and isopropanol, on retention properties has been assessed. Results indicate that HIC retention is also governed by conformational in the proteins which affect the number of accessible hydrophobic moieties.

  2. Purification of Mycobacterium bovis BCG Tokyo antigens by chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography.

    PubMed Central

    Sugden, E A; Stilwell, K; Watson, D C; Rohonczy, E B; Martineau, P

    1996-01-01

    A combination of chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography resulted in a simple purification of protein antigens of Mycobacterium bovis BCG Tokyo culture filtrate. Identification was established on the basis of chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis determination of molecular weights, and N-terminal amino acid determination. Chromatofocusing on PBE 94 accomplished the separation of BCG85B from other BCG85 complex antigens and partial separation of MPB64 and MPB70 antigens. Subsequently, MPB64 and MPB70 were completely separated on a high-performance liquid chromatography TSK Phenyl 5PW hydrophobic interaction chromatography column. This column also separated BCG85B from a 17-kDa protein with an N-terminal amino acid sequence of A-V-P-I-T-G-K-L-G-S-E-L-T-M-T-D-( )-V-G-Q, which is similar to the sequence of MPT63. Concanavalin A-Sepharose-affinity chromatography separated MPB64 from a 43- and 47-kDa doublet with an amino acid sequence of D-P-E-P-A-P-P-V-P-P-V-P-A-( )-A-A-S-P, which is similar to the sequence of MPT32 and which appears to be glycosylated. PMID:8877132

  3. Purification of Mycobacterium bovis BCG Tokyo antigens by chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography.

    PubMed

    Sugden, E A; Stilwell, K; Watson, D C; Rohonczy, E B; Martineau, P

    1996-09-01

    A combination of chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography resulted in a simple purification of protein antigens of Mycobacterium bovis BCG Tokyo culture filtrate. Identification was established on the basis of chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis determination of molecular weights, and N-terminal amino acid determination. Chromatofocusing on PBE 94 accomplished the separation of BCG85B from other BCG85 complex antigens and partial separation of MPB64 and MPB70 antigens. Subsequently, MPB64 and MPB70 were completely separated on a high-performance liquid chromatography TSK Phenyl 5PW hydrophobic interaction chromatography column. This column also separated BCG85B from a 17-kDa protein with an N-terminal amino acid sequence of A-V-P-I-T-G-K-L-G-S-E-L-T-M-T-D-( )-V-G-Q, which is similar to the sequence of MPT63. Concanavalin A-Sepharose-affinity chromatography separated MPB64 from a 43- and 47-kDa doublet with an amino acid sequence of D-P-E-P-A-P-P-V-P-P-V-P-A-( )-A-A-S-P, which is similar to the sequence of MPT32 and which appears to be glycosylated.

  4. Adsorption mechanism in reversed-phase liquid chromatography. Effect of the surface coverage of a monomeric C18-silica stationary phase

    SciTech Connect

    Gritti, Fabrice; Guiochon, Georges A

    2006-04-01

    The effect of the bonding density of the octadecyl chains onto the same silica on the adsorption and retention properties of low molecular weight compounds (phenol, caffeine, and sodium 2-naphthalene sulfonate) was investigated. The same mobile phase (methanol:water, 20:80, v/v) and temperature (T = 298 K) were applied and two duplicate columns (A and B) from each batch of packing material (neat silica, simply endcapped or C{sub 1} phase, 0.42, 1.01, 2.03, and 3.15 {micro}mol/m{sup 2} of C{sub 18} alkyl chains) were tested. Adsorption data of the three compounds were acquired by frontal analysis (FA) and the adsorption energy distributions (AEDs) were calculated using the expectation-maximization method. Results confirmed earlier findings in linear chromatography of a retention maximum at an intermediate bonding density. From a general point of view, the saturation capacity of the adsorbent tends to decrease with increasing bonding density, due to the vanishing space intercalated between the C{sub 18} bonded chains and to the decrease of the specific surface area of the stationary phase. The equilibrium constants are maximum for an intermediary bonding density ({approx}2 {micro}mol/m{sup 2}). An enthalpy-entropy compensation was found for the thermodynamic parameters of the isotherm data. Weak equilibrium constants (small {Delta}H) and high saturation capacities (large {Delta}S) were observed at low bonding densities, higher equilibrium constants and lower saturation capacities at high bonding densities, the combinations leading to similar apparent retention in RPLC. The use of a low surface coverage column is recommended for preparative purposes.

  5. A chromatographic estimate of the degree of surface heterogeneity of reversed-phase liquid chromatography packing materials II-Endcapped monomeric C18-bonded stationary phase

    SciTech Connect

    Gritti, Fabrice; Guiochon, Georges A

    2006-01-01

    In a previous report, the heterogeneity of a non-endcapped C{sub 30}-bonded stationary phase was investigated, based on the results of the measurements of the adsorption isotherms of two neutral compounds (phenol and caffeine) and two ionizable compounds (sodium naphthalene sulfonate and propranololium chloride) by frontal analysis (FA). The same method is applied here for the characterization of the surface heterogeneity of two new brands of endcapped C{sub 18}-bonded stationary phases (Gemini and Sunfire). The adsorption isotherms of the same four chemicals were measured by FA and the results confirmed by the independent calculation of the adsorption energy distribution (AED), using the expectation-maximization (EM) method. The effect of the length of the bonded alkyl chain was investigated. Shorter alkyl-bonded-chains (C{sub 18} versus C{sub 30}) and the end-capping of the silica surface contribute to decrease the surface heterogeneity under the same experimental conditions (30% methanol, 25 mM NaCl). The AEDs of phenol and caffeine are bimodal with the C{sub 18}-bonded columns while they are trimodal and quadrimodal, respectively, with a non-endcapped C{sub 30}-bonded column. The 'supersites' (adsorption energy >20 kJ/mol) found on the C{sub 30}-Prontosil column and attributed to a cation exchange mechanism completely disappear on the C{sub 18}-Gemini and C{sub 18}-Sunfire, probably because the end-capping of the silica surface eliminates most if not all the ionic interactions.

  6. Cationic Ionic Liquids Organic Ligands Based Metal-Organic Frameworks for Fabrication of Core-Shell Microspheres for Hydrophilic Interaction Liquid Chromatography.

    PubMed

    Dai, Qian; Ma, Junqian; Ma, Siqi; Wang, Shengyu; Li, Lijun; Zhu, Xianghui; Qiao, Xiaoqiang

    In this study, new metal-organic frameworks (MOFs) nanocrystals modified SiO2 core-shell microspheres were designed with cationic ionic liquids (ILs) 1,3-bis(4-carboxybutyl)imidazolium bromide (ILI) as organic ligands. By further adjustment the growth cycles, the new ILI-01@SiO2 core-shell stationary phase was facilely fabricated. The developed stationary phase was respectively characterized via element analysis, thermogravimetric analysis, scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectrometry. Because the introduction of cationic imidazolium-based ILs ILI for fabrication of the MOFs nanocrystals shell, the new stationary phase exhibits the retention mechanism of hydrophilic interaction liquid chromatography (HILIC). Many polar samples, such as amides, vitamins, nucleic acid bases, and nucleosides, were utilized to investigate the performance of the prepared ILI-01@SiO2 column. Compared to the conventional aminosilica column, the new ILI-01@SiO2 column displays high separation selectivity in a shorter separation time. Furthermore, the new ILI-01@SiO2 column was also used for detection of illegal melamine addition in the baby formula. All the above results demonstrate the new ILI-01@SiO2 core-shell stationary phase is of good potentials for high-selectivity separation the polar samples.

  7. Dielectric Interactions and the Prediction of Retention Times of Pesticides in Supercritical Fluid Chromatography with CO2

    NASA Astrophysics Data System (ADS)

    Alvarez, Guillermo A.; Baumanna, Wolfram

    2005-02-01

    A thermodynamic model for the partition of a solute (pesticide) between two immiscible phases, such as the stationary and mobile phases of supercritical fluid chromatography with CO2, is developed from first principles. A key ingredient of the model is the result of the calculation made by Liptay of the energy of interaction of a polar molecule with a dielectric continuum, which represents the solvent. The strength of the interaction between the solute and the solvent, which may be considered a measure of the solvent power, is characterized by a function g = (ɛ - 1)/(2ɛ +1), where ɛ is the dielectric constant of the medium, which is a function of the temperature T and the pressure P. Since the interactions between the nonpolar supercritical CO2 solvent and the slightly polar pesticide molecules are considered to be extremely weak, a regular solution model is appropriate from the thermodynamic point of view. At constant temperature, the model predicts a linear dependence of the logarithm of the capacity factor (lnk) of the chromatographic experiment on the function g = g(P), as the pressure is varied, with a slope which depends on the dipole moment of the solute, dispersion interactions and the size of the solute cavity in the solvent. At constant pressure, once the term containing the g (solvent interaction) factor is subtracted from lnk, a plot of the resulting term against the inverse of temperature yields the enthalpy change of transfer of the solute from the mobile (supercritical CO2) phase to the stationary (adsorbent) phase. The increase in temperature with the consequent large volume expansion of the supercritical fluid lowers its solvent strength and hence the capacity factor of the column (or solute retention time) increases. These pressure and temperature effects, predicted by the model, agree excellently with the experimental retention times of seven pesticides. Beyond a temperature of about 393 K, where the liquid solvent densities approach those of

  8. Retentivity, selectivity and thermodynamic behavior of polycyclic aromatic hydrocarbons on charge-transfer and hypercrosslinked stationary phases under conditions of normal phase high performance liquid chromatography.

    PubMed

    Jiang, Ping; Lucy, Charles A

    2016-03-11

    Charge-transfer and hypercrosslinked polystyrene phases offer retention and separation for polycyclic aromatic hydrocarbons (PAHs) and thus have potential for petroleum analysis. The size, shape and planarity selectivity for PAH standards on charge-transfer (DNAP column) and hypercrosslinked polystyrene (HC-Tol and 5HGN columns) phases are different under normal phase liquid chromatography (NPLC). The HC-Tol column behaves like a conventional NPLC column with low retention of PAHs. Retention of PAHs on the DNAP and 5HGN are strong and increases with the number of aromatic rings. The main retention mechanism is through π-π interactions and dipole-induced dipole interaction. Thermodynamics indicates that the retention mechanism of PAHs remains unchanged over the temperature range 20-60°C. In addition, on either DNAP or 5HGN column, both linear and bent PAHs are retained through the same mechanism. But DNAP possesses smaller π-π interaction and higher planarity selectivity than 5HGN for PAHs. This is suggestive that DNAP interacts with PAHs through a disordered phase arrangement, while 5HGN behaves as an ordered adsorption phase. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Physico-chemical characterization of liposomes and drug substance-liposome interactions in pharmaceutics using capillary electrophoresis and electrokinetic chromatography.

    PubMed

    Franzen, Ulrik; Østergaard, Jesper

    2012-12-07

    Liposomes are self-assembled phospholipid vesicles and have numerous research and therapeutic applications. In the pharmaceutical and biomedical sciences liposomes find use as models of biological membranes, partitioning medium and as drug carriers. The present review addresses the use of capillary electrophoresis and liposome electrokinetic chromatography for the characterization of liposomes in a pharmaceutical context. Capillary electrophoretic techniques have been used for the measurement of electrophoretic mobility, which provides information on liposome surface charge, size and membrane permeability of liposomes. The use of liposome electrokinetic chromatography and capillary electrophoresis for determination of liposome/water partitioning and characterization of drug-liposome interactions is reviewed. A number of studies indicate that capillary electrophoresis may have a role in the characterization of liposome drug delivery systems, e.g., for the investigation of encapsulation efficiency and drug leakage. The well-known characteristics of capillary electrophoresis, i.e., low sample volume requirement, high separation efficiency in aqueous media without a stationary phase, minimal sample preparation, and a high degree of automation, makes it an attractive approach in liposome research.

  10. Hydrophilic interaction chromatography of seized drugs and related compounds with sub 2 μm particle columns.

    PubMed

    Lurie, Ira S; Li, Li; Toske, Steven G

    2011-12-30

    The use of hydrophilic interaction chromatography (HILIC) with sub 2 μm particle columns for the analysis of drugs and related compounds of forensic interest is described. This technique uses a high organic/low aqueous buffered mobile phase with a polar stationary phase, and is excellent for the separation of many of the charged solutes that are found in forensic drug exhibits. In this study, HILIC is investigated for 11 solutes of forensic interest, including weak bases, weak acids, and a neutral solute. In addition, for columns containing either ethylene bridged hybrid particles with or without an amide bonded phase, the effects of acetonitrile concentration, buffer type, buffer concentration, linear velocity, and sample concentration were studied. Based on these studies, HILIC with sub 2 μm particle columns can offer highly efficient, selective, and rapid isocratic separations of drugs and related compounds of forensic interest, with excellent peak shapes and low back pressures. This is in contrast to reverse phase chromatography (RPLC), where gradient elution is usually required, which can result in extensive overlap between acidic, neutral, and basic solutes. In addition, since HILIC exhibits a much greater loading capacity than RPLC, it could be a preferred technique for drug profiling. Furthermore, because high organic content mobile phases are highly amenable to mass spectrometric detection, the use of HILIC with tandem mass spectrometric detection for the analysis of seized drugs is described.

  11. Simulation of Alpha Particles in Rotating Plasma Interacting with a Stationary Ripple

    SciTech Connect

    Abraham J. Fetterman and Nathaniel J. Fisch

    2011-01-11

    Superthermal ExB rotation can provide magnetohydrodynamic (MHD) stability and enhanced confinement to axisymmetric mirrors. However, the rotation speed has been limited by phenomena at end electrodes. A new prediction is that rotation might instead be produced using a magnetic ripple and alpha particle kinetic energy, in an extension of the alpha channeling concept. The interaction of alpha particles with the ripple results in visually interesting and practically useful orbits.

  12. Hydrophobic interaction membrane chromatography for bioseparation and responsive polymer ligands involved

    NASA Astrophysics Data System (ADS)

    Chen, Jingling; Peng, Rong; Chen, Xiaonong

    2017-09-01

    Hydrophobic interaction chromatography (HIC) is a rapid growing bioseparation technique, which separates biomolecules, such as therapeutic proteins and antibodys, based on the reversible hydrophobic interaction between immobilized hydrophobic ligands on chromatographic resin spheres and non-polar regions of solute molecule. In this review, the fundamental concepts of HIC and the factors that may affect purification efficiency of HIC is summarized, followed by the comparison of HIC with affinity chromatography and ion-exchange chromatography. Hydrophobic interaction membrane chromatography (HIMC) combines the advantages of HIC and membrane process and has showed great potential in bioseparation. For better understanding of HIMC, this review presents an overview of two main concerns about HIMC, i.e. membrane materials and hydrophobic ligands. Specifically, cellulose fiber-based membrane substrate and environment-responsive ligands are emphasized.

  13. Investigation of calcium antagonist-L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method.

    PubMed

    Du, Hui; He, Jianyu; Wang, Sicen; He, Langchong

    2010-07-01

    The dissociation equilibrium constant (K(D)) is an important affinity parameter for studying drug-receptor interactions. A vascular smooth muscle (VSM) cell membrane chromatography (CMC) method was developed for determination of the K(D) values for calcium antagonist-L-type calcium channel (L-CC) interactions. VSM cells, by means of primary culture with rat thoracic aortas, were used for preparation of the cell membrane stationary phase in the VSM/CMC model. All measurements were performed with spectrophotometric detection (237 nm) at 37 degrees C. The K(D) values obtained using frontal analysis were 3.36 x 10(-6) M for nifedipine, 1.34 x 10(-6) M for nimodipine, 6.83 x 10(-7) M for nitrendipine, 1.23 x 10(-7) M for nicardipine, 1.09 x 10(-7) M for amlodipine, and 8.51 x 10(-8) M for verapamil. This affinity rank order obtained from the VSM/CMC method had a strong positive correlation with that obtained from radioligand binding assay. The location of the binding region was examined by displacement experiments using nitrendipine as a mobile-phase additive. It was found that verapamil occupied a class of binding sites on L-CCs different from those occupied by nitrendipine. In addition, nicardipine, amlodipine, and nitrendipine had direct competition at a single common binding site. The studies showed that CMC can be applied to the investigation of drug-receptor interactions.

  14. Method development and optimization on cinchona and chiral sulfonic acid-based zwitterionic stationary phases for enantiomer separations of free amino acids by high-performance liquid chromatography.

    PubMed

    Zhang, Tong; Holder, Emilie; Franco, Pilar; Lindner, Wolfgang

    2014-10-10

    CHIRALPAK ZWIX(+) and ZWIX(-) are cinchona alkaloid-derived zwitterionic chiral stationary phases (CSPs) containing a chiral sulfonic acid motif which serves as negatively charged interaction site. They are versatile for direct enantiomer resolution of amino acids and many other ampholytic compounds by HPLC. The synergistic double ion-pairing between the zwittrionic chiral selector and the ampholyte is the basis for interaction and chiral recognition mechanisms. ZWIX(+) and ZWIX(-) type CSPs or columns behave pseudo-enantiomerically and provide the feature of reversing enantiomer elution order by column switching. In the current study, extensive experimental work was carried out with the aim of developing schemes for an efficient generic screening and proposing straightforward approaches for method optimization on these ZWIX columns. Various chromatographic parameters were investigated using a large series of diverse amino acids and analogues for the purpose. The role of methanol (MeOH) as the protic solvent in the mobile phase is confirmed to be essential. The presence of water in a low percentage is beneficial for peak shape, resolution, analysis speed, sample solubility and MS detection performance. The involvement of acetonitrile (ACN) or tetrahydrofuran (THF) can help for adjusting retention time and selectivity. Incorporation of a suitable pair of acidic-basic additives at a right ratio in the mobile phase is determinant as well for the double ion-pairing mechanism. 50 mM formic acid+25 mM diethylamine (or ammonium hydroxide) in MeOH/ACN/H₂O and in MeOH/THF/H₂O at 49:49:2 (by volume) are recommended as the starting mobile phases for method development. Some other parameters are also considered in the proposed scheme to achieve successful enantioselective or stereoselective separation of the ampholytes. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Distinction of synthetic dl-α-tocopherol from natural vitamin E (d-α-tocopherol) by reversed-phase liquid chromatography. Enhanced selectivity of a polymeric C18 stationary phase at low temperature and/or at high pressure.

    PubMed

    Yui, Yuko; Miyazaki, Shota; Ma, Yan; Ohira, Masayoshi; Fiehn, Oliver; Ikegami, Tohru; McCalley, David V; Tanaka, Nobuo

    2016-06-10

    Separation of diastereomers of dl-α-tocopherol was studied by reversed-phase liquid chromatography using three types of stationary phases, polymeric ODS, polymeric C30, and monomeric ODS. Polymeric ODS stationary phase (Inertsil ODS-P, 3mmID, 20cm) was effective for the separation of the isomers created by the presence of three chiral centers on the alkyl chain of synthetic dl-α-tocopherol. Considerable improvement of the separation of isomers was observed on ODS-P phase at high pressure and at low temperature. Complete separation of four pairs of diastereomers was achieved at 12.0°C, 536bar, while three peaks were observed when the separation was carried out either at 12.0°C at low pressure or at 20°C at 488bar. Higher temperature (30.0°C) with the ODS-P phase resulted in only partial separation of the diastereomers even at high pressure. Only slight resolution was observed for the mixture of diastereomers with the C30 stationary phase (Inertsil C30) at 12.0°C and 441bar, although the stationary phase afforded greater resolution for β- and γ-tocopherol than ODS-P. A monomeric C18 stationary phase did not show any separation at 12.0°C and 463bar. The results suggest that the binding site of the polymeric ODS-P phase is selective for flexible alkyl chains that provided the longest retention for the natural form, (R,R,R) form, and the enantiomer, (S,S,S) form, of dl-α-tocopherol.

  16. Single-step purification of bispecific monoclonal antibodies for immunotherapeutic use by hydrophobic interaction chromatography.

    PubMed

    Manzke, O; Tesch, H; Diehl, V; Bohlen, H

    1997-10-13

    A method for large scale production and single-step purification of bispecific antibodies is described. Hybrid-hybridomas were grown in hollow-fibre bioreactors with an average yield of 8 to 12 g of immunoglobulin per month. Bispecific antibodies were purified from the bioreactor supernatant by hydrophobic interaction chromatography which resolves bispecific antibodies, monospecific immunoglobulins, and culture medium supplements in one single chromatographic step. Proteins were analyzed by ELISA, SDS-PAGE, isoelectric focussing, indirect fluorescence staining, CTL-stimulation and T-cell proliferation assays. Finally, antibody preparations were checked for the presence of endotoxin and mouse DNA. Our results suggest that functional bispecific antibodies for use in therapeutic applications can be batch purified from bioreactor harvest by hydrophobic interaction chromatography in a single step. Compared to other methods such as affinity chromatography (protein A/G), ion-exchange or hydroxyapatite chromatography, our protocol offers a substantial reduction in labor time, cost, protein loss, and risk of contamination.

  17. Frontal affinity chromatography: A unique research tool for biospecific interaction that promotes glycobiology

    PubMed Central

    KASAI, Kenichi

    2014-01-01

    Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described. PMID:25169774

  18. A fast, simple, and reliable hydrophilic interaction liquid chromatography method for the determination of ascorbic and isoascorbic acids.

    PubMed

    Barros, Ana I R N A; Silva, Ana P; Gonçalves, Berta; Nunes, Fernando M

    2010-03-01

    A reliable method for the determination of total vitamin C must be able to resolve ascorbic acid (AA) and the epimeric isoascorbic acid (IAA) and determine the sum of AA and its oxidized form dehydroascorbic acid. AA and IAA are polar molecules with a low retention time in conventional reversed phase systems, and hence of difficult resolution. Hydrophilic interaction chromatography using a TSKgel Amide-80 stationary phase with isocratic elution was successful in resolving the two epimers. The column was compatible with injections of high concentrations of metaphosphoric acid, tris(2-carboxyethyl)-phosphine, and EDTA without drift of baseline and retention time. Total AA and IAA were extracted, stabilized, and reduced in one step at 40 °C, using 5% m-phosphoric acid, 2 mM of EDTA, and 2 mM of tris(2-carboxyethyl)-phosphine as reducing agent. This simple, fast, and robust hydrophilic interaction chromatography-DAD method was applied for the analysis of food products namely fruit juices, chestnut, and ham and also in pharmaceutical and multivitamin tablets. Method validation was performed on the food products, including parameters of precision, accuracy, linearity, limit of detection, and quantification (LOQ). The absence of matrix interferences was assessed by the standard addition method and Youden calibration. The method was fast, accurate, and precise with a LOQ(AA) of 1.5 mg/L and LOQ(IAA) of 3.7 mg/L. The simple experimental procedure, completed in 1 h, the possibility of using IAA as an internal standard, and low probability of artifacts are the major advantages of the proposed method for the routine determination of these compounds in a large number of samples.

  19. Multi-modal applicability of a reversed-phase/weak-anion exchange material in reversed-phase, anion-exchange, ion-exclusion, hydrophilic interaction and hydrophobic interaction chromatography modes.

    PubMed

    Lämmerhofer, Michael; Nogueira, Raquel; Lindner, Wolfgang

    2011-06-01

    We recently introduced a mixed-mode reversed-phase/weak anion-exchange type separation material based on silica particles which consisted of a hydrophobic alkyl strand with polar embedded groups (thioether and amide functionalities) and a terminal weak anion-exchange-type quinuclidine moiety. This stationary phase was designed to separate molecules by lipophilicity and charge differences and was mainly devised for peptide separations with hydroorganic reversed-phase type elution conditions. Herein, we demonstrate the extraordinary flexibility of this RP/WAX phase, in particular for peptide separations, by illustrating its applicability in various chromatographic modes. The column packed with this material can, depending on the solute character and employed elution conditions, exploit attractive or repulsive electrostatic interactions, and/or hydrophobic or hydrophilic interactions as retention and selectivity increments. As a consequence, the column can be operated in a reversed-phase mode (neutral compounds), anion-exchange mode (acidic compounds), ion-exclusion chromatography mode (cationic solutes), hydrophilic interaction chromatography mode (polar compounds), and hydrophobic interaction chromatography mode (e.g., hydrophobic peptides). Mixed-modes of these chromatographic retention principles may be materialized as well. This allows an exceptionally flexible adjustment of retention and selectivity by tuning experimental conditions. The distinct separation mechanisms will be outlined by selected examples of peptide separations in the different modes.

  20. Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms.

    PubMed

    Beres, Martin J; Olesik, Susan V

    2015-07-06

    The potential of enhanced-fluidity liquid chromatography, a subcritical chromatography technique, in mixed-mode hydrophilic interaction/strong cation-exchange separations is explored, using amino acids as analytes. The enhanced-fluidity liquid mobile phases were prepared by adding liquefied CO2 to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO2 to methanol/water mixtures resulted in increased retention of the more polar amino acids. The "optimized" chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO2 mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO2 mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced-fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced-fluidity mobile phase separation was governed by a mixed-mode retention mechanism of hydrophilic interaction/strong cation-exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Hydrophilic interaction liquid chromatography-mass spectrometry as a new tool for the characterization of intact semi-synthetic glycoproteins.

    PubMed

    Tengattini, Sara; Domínguez-Vega, Elena; Temporini, Caterina; Bavaro, Teodora; Rinaldi, Francesca; Piubelli, Luciano; Pollegioni, Loredano; Massolini, Gabriella; Somsen, Govert W

    2017-08-15

    Improved methods for detailed characterization of complex glycoproteins are required in the growing sector of biopharmaceuticals. Hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution (HR) time-of-flight mass spectrometric (TOF-MS) detection was examined for the characterization of intact neo-glycoproteins prepared by chemical conjugation of synthetic saccharides to the lysine residues of selected recombinant proteins. The separation performances of three different amide HILIC columns (TSKgel Amide-80, XBridge BEH and AdvanceBio Glycan Mapping) were tested. Water-acetonitrile gradients and volatile eluent additives have been explored. Addition of 0.05% (v/v) trifluoroacetic acid to the mobile phase appeared to be essential for achieving optimum resolution of intact glycoforms and minimal ion suppression effects. Gradient elution conditions were optimized for each protein on every column. HILIC stationary phases were evaluated for the analysis of highly heterogeneous semi-synthetic derivatives of the same protein (ribonuclease A), and in the enhanced characterization of TB10.4 and Ag85B glycoconjugates, selected antigens from Mycobacterium tuberculosis (MTB). HILIC-MS results indicated that the HILIC selectivity is predominantly governed by size of the conjugated glycans and number of glycans attached, providing efficient glycoform separation. Moreover, HILIC separation prior to HRMS detection allowed assignment of several product impurities. Additional top-down MS/MS experiments confirmed conjugation at the N-terminus of TB10.4 next to its lysine residue. Overall, the obtained results demonstrate that amide-stationary-phase based HILIC coupled to MS is highly useful for the characterization of intact neo-glycoproteins allowing assessment of the number, identity and relative abundance of glycoforms present in the semi-synthetic products. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Cucurbit(6)uril immobilized on silica: a novel high-performance liquid chromatographic stationary phase.

    PubMed

    Ma, Liyun; Liu, Simin; Wang, Qing; Yao, Lin; Xu, Li

    2015-04-01

    In the present study, one of the new generation of host molecules, cucurbit(6)uril (CB(6)), was immobilized onto silica (CB(6)/SiO2 ) by a sol-gel approach. CB(6)/SiO2 was characterized by NMR spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and elemental analysis. It was used as a high-performance liquid chromatographic stationary phase and its chromatographic performance was systematically investigated with different types of analytes as probes. The results revealed that the CB(6)/SiO2 stationary phase exhibited weak hydrophobic and strong hydrophilic properties. Hence, the variables for hydrophilic interaction liquid chromatography, including components and pH of the mobile phase, were further investigated to explore the retention mechanism of this CB(6)/SiO2 stationary phase. For less polar analytes, both hydrophobic and hydrophilic interactions could contribute to the retention, while for polar analytes, hydrophilic interaction may be predominant. Compared to the tetraethoxylsilane-coated SiO2 stationary phases, the CB(6)/SiO2 stationary phase exhibited a different retention behavior toward basic analytes with excellent stability. It is a novel promising hydrophilic interaction liquid chromatography stationary phase.

  3. Preparative isolation of flavonoid glycosides from Sphaerophysa salsula using hydrophilic interaction solid-phase extraction coupled with two-dimensional preparative liquid chromatography.

    PubMed

    Jiao, Lijin; Tao, Yanduo; Wang, Weidong; Shao, Yun; Mei, Lijuan; Wang, Qilan; Dang, Jun

    2017-07-31

    An offline preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography coupled with hydrophilic interaction solid-phase extraction method was developed for the preparative isolation of flavonoid glycosides from a crude sample of Sphaerophysa salsula. First, the non-flavonoids were removed using an XAmide solid-phase extraction cartridge. Based on the separation results of three different chromatographic stationary phases, the first-dimensional preparation was performed on an XAqua C18 prep column, and 15 fractions were obtained from the 5.2 g target sample. Then, three representative fractions were selected for additional purification on an XAmide preparative column to further isolate the flavonoid glycosides. In all, eight flavonoid glycosides were isolated in purities over 97%. The results demonstrated that the two-dimensional liquid chromatography method used in this study was effective for the preparative separation of flavonoid glycosides from Sphaerophysa salsula. Additionally, this method showed great potential for the separation of flavonoid glycosides from other plant materials. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Development and evaluation of a hydrophilic interaction liquid chromatography-MS/MS method to quantify 19 nucleobases and nucleosides in rat plasma.

    PubMed

    Du, Yan; Li, Yin-Jie; Hu, Xun-Xiu; Deng, Xu; Qian, Zeng-Ting; Li, Zheng; Guo, Meng-Zhe; Tang, Dao-Quan

    2017-04-01

    As essential endogenous compounds, nucleobases and nucleosides fulfill various functions in living organisms. This study presents the development and validation of a new hydrophilic interaction liquid chromatography tandem mass spectrometry method for simultaneous quantification of 19 nucleobases and nucleosides in rat plasma. For the sample preparation, 15 kinds of protein precipitants were evaluated according to the chromatographic profile and ion response of analytes. The optimization of chromatographic separation was respectively performed using reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode; each separation mode included two test columns with different stationary phases. The chromatographic profile and parameters such as half-width (W1/2 ), capacity factor (K') and tailing factor (ft ) were used to evaluate the separation efficiencies. Furthermore, the adopted composition of two mobile phase systems and the concentrations of the additives in the optimum buffer system were also investigated. The developed method was fully validated and successfully applied quantitatively to determine 19 nucleobases and nucleosides in plasma from normal and diabetic nephropathy (DN) rats. Significant differences between normal and DN rats were found in plasma levels of cytosine, xanthine, thymidine, adenosine, guanosine, inosine and 8-hydroxy-2'-deoxyguanosine. This information may provide a useful reference for the discovery of potential biomarkers of DN. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Protein-surface interaction maps for ion-exchange chromatography.

    PubMed

    Freed, Alexander S; Cramer, Steven M

    2011-04-05

    In this paper, protein-surface interaction maps were generated by performing coarse-grained protein-surface calculations. This approach allowed for the rapid determination of the protein-surface interaction energies at a range of orientations and distances. Interaction maps of lysozyme indicated that there was a contiguous series of orientations corresponding to several adjacent preferred binding regions on the protein surface. Examination of these orientations provided insight into the residues involved in surface interactions, which qualitatively agreed with the retention data for single-site mutants. Interaction maps of lysozyme single-site mutants were also generated and provided significant insight into why these variants exhibited significant differences in their chromatographic behavior. This approach was also employed to study the binding behavior of CspB and related mutants. The results indicated that, in addition to describing general trends in the data, these maps provided significant insight into retention data of the single-site mutants. In particular, subtle retention trends observed with the K12 and K13 mutants were well-described using this interaction map approach. Finally, the number of interaction points with energies stronger than -2 kcal/mol was shown to be able to semi-quantitatively predict the behavior of most of the mutants. This rapid approach for calculating protein-surface interaction maps is expected to facilitate future method development for separating closely related protein variants in ion-exchange systems.

  6. Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications.

    PubMed

    Hage, David S

    2017-06-01

    The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically related binding agent, are 2 methods that can be used to study these interactions. This review presents various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent. The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples. © 2016 American Association for Clinical Chemistry.

  7. Two variables dominating the retention of intact proteins under gradient elution with simultaneous ultrafast high-resolution separation by hydrophobic interaction chromatography.

    PubMed

    Geng, Xindu; Jia, Xiaodan; Liu, Peng; Wang, Fei; Yang, Xiaoming

    2015-10-07

    The retention of intact proteins under gradient elution in hydrophobic interaction chromatography (HIC) was found to be governed by two variables, the steady region (SR) and the migration region (MR). In the SR, the proteins are immobilized by the strong interactions with the stationary phase such that the retention time is independent of the column length. In the MR, the proteins also interact with the stationary phase, but they move normally, thus the retention time depends on their partition coefficients and the column length. The SR can be used as an operation space (OP) for high-throughput protein analysis by 1D-LC using short columns at high flow rates to maintain a high resolution. The OP can also be employed for all assisted operations in online 2D-LC. Based on the steady region/migration region optimization strategy developed in this study, five successive complete separations of seven intact proteins were performed in a HIC cake in less than 5 min, and a crude extract of ribonuclease A from bovine pancreas was purified using online 2D-LC to 95.8% purity with 93.2% mass recovery in 45 min. This approach can be used to expedite the purification of drug-target proteins and should therefore be of interest to the pharmaceutical industry.

  8. Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography.

    PubMed

    Tran, Benjamin; Grosskopf, Vanessa; Wang, Xiangdan; Yang, Jihong; Walker, Don; Yu, Christopher; McDonald, Paul

    2016-03-18

    Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry to determine artificial sweeteners in environmental waters.

    PubMed

    Salas, Daniela; Borrull, Francesc; Fontanals, Núria; Marcé, Rosa Maria

    2015-06-01

    Artificial sweeteners are food additives employed as sugar substitutes which are now considered to be emerging organic contaminants. In the present study, a method is developed for the determination of a group of artificial sweeteners in environmental waters. Considering the polar and hydrophilic character of these compounds, hydrophilic interaction liquid chromatography is proposed for their separation as an alternative to traditional reversed-phase liquid chromatography. Two stationary phases with different chemistry were compared for this purpose. For the detection of the analytes, high-resolution mass spectrometry (Orbitrap) was employed to take advantage of its benefits in terms of reliable quantification and confirmation for the measurement of accurate masses. Solid-phase extraction was chosen as the sample treatment, in which the extract in a mixture of NH4OH:MeOH:ACN (1:4:15) was directly injected into the chromatographic system, simplifying the analytical procedure. The optimized method was validated on river and waste water samples. For example, in the case of effluent water samples, limits of detection ranged from 0.002 to 0.7 μg/L and limits of quantification ranged from 0.004 to 1.5 μg/L. Apparent (whole method) recoveries ranged from 57 to 74% with intra-day precision (%RSD, n = 5) ranging from 6 to 25%. The method was successfully applied to water samples from different rivers in Catalonia and different waste water treatment plants in Tarragona. Acesulfame, cyclamate, saccharine and sucralose were found in several samples.

  10. Direct measurement of protein osmotic second virial cross coefficients by cross-interaction chromatography.

    PubMed

    Tessier, Peter M; Sandler, Stanley I; Lenhoff, Abraham M

    2004-05-01

    The importance of weak protein interactions, such as protein self-association, is widely recognized in a variety of biological and technological processes. Although protein self-association has been studied extensively, much less attention has been devoted to weak protein cross-association, mainly due to the difficulties in measuring weak interactions between different proteins in solution. Here a framework is presented for quantifying the osmotic second virial cross coefficient directly using a modified form of self-interaction chromatography called cross-interaction chromatography. A theoretical relationship is developed between the virial cross coefficient and the chromatographic retention using statistical mechanics. Measurements of bovine serum albumin (BSA)/lysozyme cross-association using cross-interaction chromatography agree well with the few osmometry measurements available in the literature. Lysozyme/alpha-chymotrypsinogen interactions were also measured over a wide range of solution conditions, and some counterintuitive trends were observed that may provide new insight into the molecular origins of weak protein interactions. The virial cross coefficients presented in this work may also provide insight into separation processes that are influenced by protein cross-interactions, such as crystallization, precipitation, and ultrafiltration.

  11. Direct measurement of protein osmotic second virial cross coefficients by cross-interaction chromatography

    PubMed Central

    Tessier, Peter M.; Sandler, Stanley I.; Lenhoff, Abraham M.

    2004-01-01

    The importance of weak protein interactions, such as protein self-association, is widely recognized in a variety of biological and technological processes. Although protein self-association has been studied extensively, much less attention has been devoted to weak protein cross-association, mainly due to the difficulties in measuring weak interactions between different proteins in solution. Here a framework is presented for quantifying the osmotic second virial cross coefficient directly using a modified form of self-interaction chromatography called cross-interaction chromatography. A theoretical relationship is developed between the virial cross coefficient and the chromatographic retention using statistical mechanics. Measurements of bovine serum albumin (BSA)/lysozyme cross-association using cross-interaction chromatography agree well with the few osmometry measurements available in the literature. Lysozyme/α-chymotrypsinogen interactions were also measured over a wide range of solution conditions, and some counterintuitive trends were observed that may provide new insight into the molecular origins of weak protein interactions. The virial cross coefficients presented in this work may also provide insight into separation processes that are influenced by protein cross-interactions, such as crystallization, precipitation, and ultrafiltration. PMID:15075404

  12. Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

    PubMed

    Brgles, Marija; Kurtović, Tihana; Kovačič, Lidija; Križaj, Igor; Barut, Miloš; Lang Balija, Maja; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

  13. Monolithic stationary phases with incorporated fumed silica nanoparticles. Part II. Polymethacrylate-based monolithic column with "covalently" incorporated modified octadecyl fumed silica nanoparticles for reversed-phase chromatography.

    PubMed

    Aydoğan, Cemil; El Rassi, Ziad

    2016-05-06

    This study is concerned with the incorporation of surface modified fumed silica nanoparticles (FSNPs) into polymethacrylate based monolithic columns for use in reversed phase chromatography (RPC) of small solutes and proteins. First, FSNPs were modified with 3-(trimethoxysilyl)propylmethacrylate (TMSPM) to yield the "hybrid" methacryloyl fumed silica nanoparticle (MFSNP) monomer. The resulting MFSNP was then mixed with glyceryl monomethacrylate (GMM) and ethylene dimethacrylate (EDMA) in a binary porogenic solvent composed of cyclohexanol and dodecanol, and the in situ copolymerization of MFSNP, GMM and EDMA was performed in a stainless steel column of 4.6 mm i.d. The silanol groups of the hybrid monolith thus obtained were grafted with octadecyl ligands by perfusing the hybrid monolithic column with a solution of 4% w/v of dimethyloctadecylchlorosilane (DODCS) in toluene while the column was maintained at 110°C for 6h (in a heated HPLC oven). One of the originalities of this study was to demonstrate MFSNP as a novel derivatized "hybrid monomer" in making RPC monolithic columns with surface bound octadecyl ligands. In this respect, the RPC behavior of the monolithic column with "covalently" incorporated FNSPs having surface grafted octadecyl ligands was evaluated with alkylbenzenes, aniline derivatives and phenolic compounds. The results showed that the hybrid poly(GMA-EDMA-MFSNP) having surface bound octadecyl ligands exhibited hydrophobic interactions under reversed phase elution conditions. Furthermore, six standard proteins were baseline separated on the column using a 10min linear gradient elution at increasing ACN concentration in the mobile phase at a flow rate of 1.0mL/min using a 10 cm×4.6mm i.d. column. The relative standard deviations (RSDs) for the retention times of the tested solutes were lower than 2.1% and 2.4% under isocratic elution and gradient elution conditions, respectively.

  14. Silica-based monolithic capillary columns modified by liposomes for characterization of analyte-liposome interactions by capillary liquid chromatography.

    PubMed

    Moravcová, Dana; Planeta, Josef; Wiedmer, Susanne K

    2013-11-22

    This study introduces a silica-based monolith in a capillary format (0.1 mm × 100 mm) as a support for immobilization of liposomes and its characterization in immobilized liposome chromatography. Silica-based monolithic capillary columns prepared by acidic hydrolysis of tetramethoxysilane in the presence of polyethylene glycol and urea were modified by (3-aminopropyl)trimethoxysilane, whereby amino groups were introduced to the monolithic surface. These groups undergo reaction with glutaraldehyde to form an iminoaldehyde, allowing covalent binding of pre-formed liposomes containing primary amino groups. Two types of phospholipid vesicles were used for column modification; these were 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidyl choline with and without 1,2-diacyl-sn-glycero-3-phospho-L-serine. The prepared columns were evaluated under isocratic separation conditions employing 20mM phosphate buffer at pH 7.4 as a mobile phase and a set of unrelated drugs as model analytes. The liposome layer on the synthesized columns significantly changed the column selectivity compared to the aminopropylsilylated monolithic stationary phase. Monolithic columns modified by liposomes were stable under the separation conditions, which proved the applicability of the suggested preparation procedure for the synthesis of capillary columns dedicated to study analyte-liposome interactions. The column efficiency originating from the silica monolith was preserved and reached, e.g., more than 120,000 theoretical plates/m for caffeine as a solute.

  15. Hydrophilic interaction liquid chromatography-mass spectrometry of (lyso)phosphatidic acids, (lyso)phosphatidylserines and other lipid classes.

    PubMed

    Cífková, Eva; Hájek, Roman; Lísa, Miroslav; Holčapek, Michal

    2016-03-25

    The goal of this work is a systematic optimization of hydrophilic interaction liquid chromatography (HILIC) separation of acidic lipid classes (namely phosphatidic acids-PA, lysophosphatidic acids-LPA, phosphatidylserines-PS and lysophosphatidylserines-LPS) and other lipid classes under mass spectrometry (MS) compatible conditions. The main parameters included in this optimization are the type of stationary phases used in HILIC, pH of the mobile phase, the type and concentration of mobile phase additives. Nine HILIC columns with different chemistries (unmodified silica, modified silica using diol, 2-picolylamine, diethylamine and 1-aminoanthracene and hydride silica) are compared with the emphasis on peak shapes of acidic lipid classes. The optimization of pH is correlated with the theoretical calculation of acidobasic equilibria of studied lipid classes. The final method using the hydride column, pH 4 adjusted by formic acid and the gradient of acetonitrile and 40 mmol/L of aqueous ammonium formate provides good peak shapes for all analyzed lipid classes including acidic lipids. This method is applied for the identification of lipids in real samples of porcine brain and kidney extracts.

  16. Hybrid carbon nanoparticles modified core-shell silica: a high efficiency carbon-based phase for hydrophilic interaction liquid chromatography.

    PubMed

    Ibrahim, Mohammed E A; Wahab, M Farooq; Lucy, Charles A

    2014-04-11

    Hydrophilic interaction liquid chromatography (HILIC) is a fast growing separation technique for hydrophilic and polar analytes. In this work, we combine the unique selectivity of carbon surfaces with the high efficiency of core-shell silica. First, 5 μm core-shell silica is electrostatically coated with 105 nm cationic latex bearing quaternary ammonium groups. Then 50 nm anionic carbon nanoparticles are anchored onto the surface of the latex coated core-shell silica particles to produce a hybrid carbon-silica phase. The hybrid phase shows different selectivity than ten previously classified HILIC column chemistries and 36 stationary phases. The hybrid HILIC phase has shape selectivity for positional isomeric pairs (phthalic/isophthalic and 1-naphthoic/2-naphthoic acids). Fast and high efficiency HILIC separations of biologically important carboxylates, phenols and pharmaceuticals are reported with efficiencies up to 85,000 plates m(-1). Reduced plate height of 1.9 (95,000 plates m(-1)) can be achieved. The hybrid phase is stable for at least 3 months of usage and storage under typical HILIC eluents. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. System maps for retention of small neutral compounds on a biphenylsiloxane-bonded silica stationary phase in reversed-phase liquid chromatography.

    PubMed

    Atapattu, Sanka N; Poole, Colin F; Praseuth, Mike B

    2016-12-23

    The system constants of the solvation parameter model are used to prepare system maps for the retention of small neutral compounds on a biphenylsiloxane-bonded superficially porous silica stationary phase (Kinetex Biphenyl) for aqueous-organic solvent mobile phases containing 10-70% (v/v) methanol or acetonitrile. The retention properties of the biphenylsiloxane-bonded phase are shown to be complementary to an octadecylsiloxane-bonded silica (Kinetex C-18) and a pentafluorophenylpropylsiloxane-bonded silica stationary phases (Discovery HS F5). The retention properties of the Kinetex Biphenyl column are similar to an ether-linked phenylpropylsiloxane-bonded silica phase (Synergi Polar RP) with only small differences in relative retention. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography.

    PubMed Central

    Tessier, Peter M; Lenhoff, Abraham M; Sandler, Stanley I

    2002-01-01

    Weak protein interactions are often characterized in terms of the osmotic second virial coefficient (B(22)), which has been shown to correlate with protein phase behavior, such as crystallization. Traditional methods for measuring B(22), such as static light scattering, are too expensive in terms of both time and protein to allow extensive exploration of the effects of solution conditions on B(22). In this work we have measured protein interactions using self-interaction chromatography, in which protein is immobilized on chromatographic particles and the retention of the same protein is measured in isocratic elution. The relative retention of the protein reflects the average protein interactions, which we have related to the second virial coefficient via statistical mechanics. We obtain quantitative agreement between virial coefficients measured by self-interaction chromatography and traditional characterization methods for both lysozyme and chymotrypsinogen over a wide range of pH and ionic strengths, yet self-interaction chromatography requires at least an order of magnitude less time and protein than other methods. The method thus holds significant promise for the characterization of protein interactions requiring only commonly available laboratory equipment, little specialized expertise, and relatively small investments of both time and protein. PMID:11867474

  19. Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography.

    PubMed

    Tessier, Peter M; Lenhoff, Abraham M; Sandler, Stanley I

    2002-03-01

    Weak protein interactions are often characterized in terms of the osmotic second virial coefficient (B(22)), which has been shown to correlate with protein phase behavior, such as crystallization. Traditional methods for measuring B(22), such as static light scattering, are too expensive in terms of both time and protein to allow extensive exploration of the effects of solution conditions on B(22). In this work we have measured protein interactions using self-interaction chromatography, in which protein is immobilized on chromatographic particles and the retention of the same protein is measured in isocratic elution. The relative retention of the protein reflects the average protein interactions, which we have related to the second virial coefficient via statistical mechanics. We obtain quantitative agreement between virial coefficients measured by self-interaction chromatography and traditional characterization methods for both lysozyme and chymotrypsinogen over a wide range of pH and ionic strengths, yet self-interaction chromatography requires at least an order of magnitude less time and protein than other methods. The method thus holds significant promise for the characterization of protein interactions requiring only commonly available laboratory equipment, little specialized expertise, and relatively small investments of both time and protein.

  20. Comprehensive characterization of Stevia rebaudiana using two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography.

    PubMed

    Fu, Qing; Guo, Zhimou; Zhang, Xiuli; Liu, Yanfang; Liang, Xinmiao

    2012-07-01

    Two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography (2D-RPLC/HILIC) system was successfully applied for comprehensive characterization of steviol glycosides from Stevia rebaudiana. The experiments were performed in offline mode using an XCharge C18 column in first dimension and an XAmide column in second dimension. In first dimension, preliminary separation of Stevia aqueous extract was accomplished and 30 fractions were collected. Then fractions 1-20 were selected for further purification and 13 compounds with high purity were obtained in second dimension. Comprehensive characterization of these compounds was completed by determination of their retention time, accurate molecular weight, diagnostic fragmentation ions, and nuclear magnetic resonance spectroscopy. As a result, all nine known steviol glycosides, as well as other four steviol glycosides were fully purified. The result demonstrated that this procedure is an effective approach for the preparative separation and comprehensive characterization of steviol glycosides in Stevia. This 2D-RPLC/HILIC method will be a promising tool for the purification of low-abundance compounds from natural products.

  1. Effect of pressure on the selectivity of polymeric C18 and C30 stationary phases in reversed-phase liquid chromatography. Increased separation of isomeric fatty acid methyl esters, triacylglycerols, and tocopherols at high pressure.

    PubMed

    Okusa, Kensuke; Iwasaki, Yuki; Kuroda, Ikuma; Miwa, Shohei; Ohira, Masayoshi; Nagai, Toshiharu; Mizobe, Hoyo; Gotoh, Naohiro; Ikegami, Tohru; McCalley, David V; Tanaka, Nobuo

    2014-04-25

    A high-density, polymeric C18 stationary phase (Inertsil ODS-P) or a polymeric C30 phase (Inertsil C30) provided improved resolution of the isomeric fatty acids (FAs), FA methyl esters (FAMEs), triacylglycerols (TAGs), and tocopherols with an increase in pressure of 20-70MPa in reversed-phase HPLC. With respect to isomeric C18 FAMEs with one cis-double bond, ODS-P phase was effective for recognizing the position of a double bond among petroselinic (methyl 6Z-octadecenoate), oleic (methyl 9Z-octadecenoate), and cis-vaccenic (methyl 11Z-octadecenoate), especially at high pressure, but the differentiation between oleic and cis-vaccenic was not achieved by C30 phase regardless of the pressure. A monomeric C18 phase (InertSustain C18) was not effective for recognizing the position of the double bond in monounsaturated FAME, while the separation of cis- and trans-isomers was achieved by any of the stationary phases. The ODS-P and C30 phases provided increased separation for TAGs and β- and γ-tocopherols at high pressure. The transfer of FA, FAME, or TAG molecules from the mobile phase to the ODS-P stationary phase was accompanied by large volume reduction (-30∼-90mL/mol) resulting in a large increase in retention (up to 100% for an increase of 50MPa) and improved isomer separation at high pressure. For some isomer pairs, the ODS-P and C30 provided the opposite elution order, and in each case higher pressure improved the separation. The two stationary phases showed selectivity for the isomers having rigid structures, but only the ODS-P was effective for differentiating the position of a double bond in monounsaturated FAMEs. The results indicate that the improved isomer separation was provided by the increased dispersion interactions between the solute and the binding site of the stationary phase at high pressure.

  2. Determination of 1-deoxynojirimycin in mulberry leaves using hydrophilic interaction chromatography with evaporative light scattering detection.

    PubMed

    Kimura, Toshiyuki; Nakagawa, Kiyotaka; Saito, Yuko; Yamagishi, Kenji; Suzuki, Masahiro; Yamaki, Kohji; Shinmoto, Hiroshi; Miyazawa, Teruo

    2004-03-24

    A simple and rapid method for determining 1-deoxynojirimycin (DNJ), a potent glucosidase inihibitor present in mulberry leaves (Morus alba and Morus bombysis), by high-performance liquid chromatography coupled to an evaporative light scattering detector (ELSD) has been developed. DNJ was separated from an extract of mulberry leaves on a TSKgel Amide-80 column, which is a representative column for hydrophilic interaction chromatography. During postcolumn detection, DNJ was detected by ELSD and concurrently identified by mass spectrometry. The detection limit was 100 ng. This method is sufficiently sensitive for determining DNJ in mulberry leaves and other related products.

  3. Quantitative analysis of oleic acid and three types of polyethers according to the number of hydroxy end groups in Polysorbate 80 by hydrophilic interaction chromatography at critical conditions.

    PubMed

    Zhang, Rui; Wang, Yu; Ji, Yu; Shi, Bei-jia; Zhang, Zai-ping; Zhang, Hai-yan; Yang, Ming; Wang, Yong-mei

    2013-01-11

    A quantitative characterization of Polysorbate 80 is crucial for its many applications. In this paper we report a quick RP-HPLC method for the quantitative determination of Polysorbate 80. The hydrolysis of Polysorbate 80 to release oleic acid and three types of polyethers was first carried out. A chromatographic method based on liquid chromatography at critical conditions (LCCC) was then developed for an endgroup-based separation of low-molecular-mass polyether. With this method the polyether, irrespective of its molecular-mass, is separated according to endgroups (functionality) due to interactions of the polar endgroups with the hydrophilic stationary phase. The different types of polyethers and oleic acid were identified using on-line electrospray ionization mass spectrometry and quantified by evaporative light scattering detection.

  4. Fully automatable two-dimensional hydrophilic interaction liquid chromatography-reversed phase liquid chromatography with online tandem mass spectrometry for shotgun proteomics.

    PubMed

    Zhao, Yun; Kong, Ricky P W; Li, Guohui; Lam, Maggie P Y; Law, C H; Lee, Simon M Y; Lam, Herman C; Chu, Ivan K

    2012-07-01

    We have developed a fully automatable two-dimensional liquid chromatography platform for shotgun proteomics analyses based on the online coupling of hydrophilic interaction liquid chromatography (HILIC) - using a nonionic type of TSKgel Amide 80 at either pH 6.8 (neutral) or 2.7 (acidic) - with conventional low-pH reversed-phase chromatography. Online coupling of the neutral-pH HILIC and reversed phase chromatography systems outperformed the acidic HILIC-reversed phase chromatography combination, resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172 unique peptides) increases in the number of identified peptides and proteins from duplicate analyses of Rat pheochromocytoma lysates. Armed with this optimized HILIC-reversed phase liquid chromatography platform, we identified 2554 nonredundant proteins from duplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately 41 to 10(6) copies per cell, which contained up to approximately 2092 different validated protein species with a dynamic range of concentrations of up to approximately 10(4) . This present study establishes a fully automated platform as a promising methodology to enable online coupling of different hydrophilic HILIC and reversed phase chromatography systems, thereby expanding the repertoire of multidimensional liquid chromatography for shotgun proteomics.

  5. Effects of the dynamic modification of stationary phases by sorbates in gas chromatography: The possibility of separating enantiomers in achiral systems

    NASA Astrophysics Data System (ADS)

    Zenkevich, I. G.; Pavlovskii, A. A.

    2016-10-01

    It is shown that the gas chromatographic separation of enantiomers on columns with achiral nonpolar stationary phases is principally possible as a result of the dynamic modification of stationary phases by sorbates under analysis. It is found that a number of key characteristic features is intrinsic to such separation: it can be only partial, it does not occur for all chromatographic columns, and it is observed only for some compounds and only within narrow ranges of quantities of sorbates that are close to the limits of mass overload of chromatographic systems. These characteristic features are illustrated by the examples of separating (1 R,5 R)-(+)- and (1 S,5 S)-(-)-α-pinenes on a WCOT column with an RTX-5 phase. The main characteristic feature of the separation of enantiomers as a result of the dynamic modification of stationary phases is the nonconformity of peaks in chromatograms with two individual enantiomers, compared to other ways and means for their separation; the first eluting peak belongs to the enantiomer that predominates in a mixture irrespective of its configuration, while the second peak corresponds to the racemic mixture of enantiomers; i.e., the ratio of peak areas in chromatograms does not correspond to the actual ratio of enantiomers in samples under analysis and is strongly distorted as a result of their incomplete separation. It is concluded that the separation of racemic mixtures in achiral systems is fundamentally impossible under any conditions, and this is one of the key criteria of the validity of the considered concept as a whole.

  6. High-performance liquid chromatography study of the enantiomer separation of chrysanthemic acid and its analogous compounds on a terguride-based stationary phase.

    PubMed

    Dondi, M; Flieger, M; Olsovska, J; Polcaro, C M; Sinibaldi, M

    1999-10-29

    The direct enantioseparation of chrysanthemic acid [2,2-dimethyl-3-(2-methylpropenyl)-cyclopropanecarboxylic acid] and its halogen-substituted analogues was systematically studied by HPLC using a terguride-based chiral stationary phase in combination with a UV diode array and chiroptical detectors. Isomers with (1R) configuration always eluted before those with (IS) configuration. The elution sequence of cis- and trans-isomers was strongly affected by mobile phase pH, whereas the enantioselectivity remained the same. Conditions for the separation of all the enantiomers were also examined. This method was used for monitor the hydrolytic degradation products of Cyfluthrin (Baythroid) in soil under laboratory conditions.

  7. Separation of polar mushroom toxins by mixed-mode hydrophilic and ionic interaction liquid chromatography-electrospray ionization-mass spectrometry.

    PubMed

    Chung, Wai-cheung; Tso, Sau-ching; Sze, Sai-tim

    2007-02-01

    Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile contaminants and naturally occurring toxins in foods. However, polar compounds, such as hydrophilic polypeptides and quaternary ammonium salts, are often not satisfactorily separated by RPLC and present a challenge for analytical scientists. In this study, hydrophilic interaction liquid chromatography (HILIC), on an amide-based stationary phase in combination with electrospray ionization (ESI) tandem mass spectrometry (MS-MS), is successfully employed to simultaneously separate polar mushroom toxins, including amanitins and phallotoxins, which are cyclic oligopeptides and muscarine, a quaternary ammonium compound, in mushrooms. The sensitivity of different ionization modes is studied, and the positive ionization mode is found to provide a more sensitive and effective tool for the unambiguous identification of the concerned polar toxins because of their characteristic fragmentation patterns. The properties of the mobile phase are also found to have significant impacts on the separation. At a high acetonitrile (ACN) concentration, hydrophilic interaction dominates, and all analytes under study demonstrate a much higher affinity with the stationary phase. The addition of methanol (MeOH) as a modifier could further enhance the HILIC separation for amanitins, phallotoxins, and muscarine. Valley-to-valley separation is achieved upon the optimatizatiqn of the mobile phase (comprising of ACN, MeOH, and ammonium formate buffer at pH approximately 3.5) and the solvent gradient. HILIC coupled with ESI-MS-MS is demonstrated to be a novel technique for the simultaneous separation and confirmatory analysis of the concerned polar toxins by providing an environment of solubility and retention that could not be achieved through the use of RPLC.

  8. Studies of a pyridino-crown ether-based chiral stationary phase on the enantioseparation of biogenic chiral aralkylamines and α-amino acid esters by high-performance liquid chromatography.

    PubMed

    Lévai, Sándor; Németh, Tamás; Fődi, Tamás; Kupai, József; Tóth, Tünde; Huszthy, Péter; Balogh, György Tibor

    2015-11-10

    This paper reports the enantioseparation ability of a pyridino-18-crown-6 ether-based chiral stationary phase [(S,S)-CSP-1]. The enantiomeric discrimination of chiral stationary phase (S,S)-CSP-1 was evaluated by HPLC using the mixtures of enantiomers of various protonated primary aralkylamines [1-phenylethylamine hydrogen perchlorate (PEA), 2,3-dihydro-1H-inden-1-amine (1-aminoindan), 2,2'-(1,2-diaminoethane-1,2-diyl) diphenol (HPEN)] and perchlorate salts of α-amino acid esters [alanine benzyl ester (Ala-OBn), phenylalanine benzyl ester (Phe-OBn), phenylalanine methyl ester (Phe-OMe), phenylglycine methyl ester (PhGly-OMe), glutamic acid dibenzyl ester (Glu-diOBn), and valine benzyl ester (Val-OBn)]. The best enantioseparation was achieved in the case of PEA. The high enantioselectivity was rationalized by the strong π-π interaction of the extended π system of the aryl-substituted pyridine unit.

  9. Development of an achiral supercritical fluid chromatography method with ultraviolet absorbance and mass spectrometric detection for impurity profiling of drug candidates. Part II. Selection of an orthogonal set of stationary phases.

    PubMed

    Lemasson, Elise; Bertin, Sophie; Hennig, Philippe; Boiteux, Hélène; Lesellier, Eric; West, Caroline

    2015-08-21

    Impurity profiling of organic products that are synthesized as possible drug candidates requires complementary analytical methods to ensure that all impurities are identified. Supercritical fluid chromatography (SFC) is a very useful tool to achieve this objective, as an adequate selection of stationary phases can provide orthogonal separations so as to maximize the chances to see all impurities. In this series of papers, we have developed a method for achiral SFC-MS profiling of drug candidates, based on a selection of 160 analytes issued from Servier Research Laboratories. In the first part of this study, focusing on mobile phase selection, a gradient elution with carbon dioxide and methanol comprising 2% water and 20mM ammonium acetate proved to be the best in terms of chromatographic performance, while also providing good MS response [1]. The objective of this second part was the selection of an orthogonal set of ultra-high performance stationary phases, that was carried out in two steps. Firstly, a reduced set of analytes (20) was used to screen 23 columns. The columns selected were all 1.7-2.5μm fully porous or 2.6-2.7μm superficially porous particles, with a variety of stationary phase chemistries. Derringer desirability functions were used to rank the columns according to retention window, column efficiency evaluated with peak width of selected analytes, and the proportion of analytes successfully eluted with good peak shapes. The columns providing the worst performances were thus eliminated and a shorter selection of columns (11) was obtained. Secondly, based on 160 tested analytes, the 11 columns were ranked again. The retention data obtained on these columns were then compared to define a reduced set of the best columns providing the greatest orthogonality, to maximize the chances to see all impurities within a limited number of runs. Two high-performance columns were thus selected: ACQUITY UPC(2) HSS C18 SB and Nucleoshell HILIC.

  10. Detailed insights into the retention mechanism of caffeine metabolites on the amide stationary phase in hydrophilic interaction chromatography.

    PubMed

    Guo, Yong; Shah, Rajan

    2016-09-09

    The amide phase was investigated using a wide range of acetonitrile content in the mobile phase in both the HILIC and RPLC modes. Using caffeine metabolites as the model compounds, the retention, thermodynamic and kinetic data was obtained under various mobile phase conditions and supported the previous postulation that there might be a transition of the predominant retention mechanism in relation to the acetonitrile content in HILIC. On the amide phase, hydrophilic partitioning seemed to be the predominant retention mechanism below 85% acetonitrile; and a different retention mechanism (presumably surface adsorption) made more and more significant contributions to the overall retention when the acetonitrile content reached above 85%. This study also provided more direct evidences to explain the effect of salt concentration on the retention of non-charged solutes in HILIC. In addition, the retention, thermodynamic and kinetic data suggest that the amide phase behaved very differently from the conventional C18 phase in the RPLC mode.

  11. Application of zirconium-modified silica gel as a stationary phase in the ion-exclusion chromatography of carboxylic acids. II. Separation of aliphatic carboxylic acids with pyromellitic acid as eluent and with suppressed conductimetric detection.

    PubMed

    Ohta, K

    2001-06-22

    The application of zirconium-modified silica gels (Zr-Silica) as stationary phases for ion-exclusion chromatography with conductimetric detection (IEC-CD) for C1-C8 aliphatic carboxylic acids (formic, acetic, propionic, butyric, valeric, caproic, heptanoic and caprylic acids) was carried out using pyromellitic acid as the eluent. Zr-Silicas were prepared by the reaction of the silanol group on the surface of silica gel with zirconium tetrabutoxide [Zr(OCH2CH2CH2CH3)4] in ethanol solution. An ASRS-Ultra anion self-regenerating suppressor in the K+ form was used for the enhancement of conductimetric detector response of these aliphatic carboxylic acids. A Zr-Silica adsorbed on 10 mg zirconium g(-1) silica gel was the most suitable stationary phase in IEC-CD for the separation of these aliphatic carboxylic acids. Excellent simultaneous separation and highly sensitive detection for these aliphatic carboxylic acids were achieved in 25 min by IEC-CD with the Zr-Silica column (250x4.6 mm I.D.) and a 0.2 mM pyromellitic acid containing 0.15% heptanol as the eluent.

  12. Application of zirconium-modified silica gel as a stationary phase in the ion-exclusion chromatography of carboxylic acids. I. Separation of benzenecarboxylic acids with tartaric acid as eluent and with UV-photometric detection.

    PubMed

    Ohta, K

    2001-06-22

    The application of zirconium-modified silica gels (Zr-Silicas) as stationary phases for ion-exclusion chromatography with UV-photometric detection (IEC-PD) for mono-, di-, tri- and tetrabenzenecarboxylic acids (pyromellitic, trimellitic, hemimellitic, o-phthalic, salicylic and benzoic acids) and phenol was carried out using tartaric acid as the eluent. Zr-Silicas were prepared by the reaction of the silanol group on the surface of silica gel with zirconium tetrabutoxide [Zr(OCH2CH2CH2CH3)4] in ethanol solution. The effect of the amount of zirconium adsorbed on silica gel on chromatographic behavior of these benzenecarboxylic acids and phenol was investigated. As a result, Zr-Silica adsorbed on 20 mg zirconium g(-1) silica gel was the most suitable stationary phase in the IEC-PD for the simultaneous separation of these benzenecarboxylic acids and phenol. Excellent simultaneous separation and highly sensitive UV detection at 254 nm for these benzenecarboxylic acids and phenol were achieved in 20 min by the IEC-PD using the Zr-Silica column (250x4.6 mm I.D.) and a 10 mM tartaric acid at pH 2.5 as eluent.

  13. Novel highly hydrophilic zwitterionic monolithic column for hydrophilic interaction chromatography.

    PubMed

    Jiang, Zhengjin; Smith, Norman W; Ferguson, Paul D; Taylor, Mark R

    2009-08-01

    A novel zwitterionic hydrophilic porous poly(SPV-co-MBA) monolithic column was prepared by thermal co-polymerisation of 1-(3-sulphopropyl)-4-vinylpyridinium-betaine (4-SPV) and N,N'-methylenebisacrylamide (MBA). An HILIC/RP dual separation mechanism was observed on this optimised poly(SPV-co-MBA) monolithic column and the composition of the mobile phase corresponding to the transition from the HILIC to the RP mode was around 30% ACN in water. Higher hydrophilicity was achieved on this novel monolithic column compared to the poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulphopropyl)ammonium betaine-co-ethylene dimethacrylate) monolithic column. Permeability studies showed slight swelling and/or shrinking with mobile phases of different polarity. As might be anticipated, a weak electrostatic interaction for charged analytes was also observed by studying the influence of mobile phase pH and salt concentration on their retention on the poly(SPV-co-MBA) monolithic column. The final optimised poly(SPV-co-MBA) monolith showed comparable selectivities to commercial ZIC-pHILIC phases for polar test analytes. Fast separation of five pyrimidines and purines was achieved in less than 1 min due to the high permeability of the monolithic column. Additionally, baseline separation of nine benzoic acid derivatives was also observed using either a pH or ACN gradient.

  14. [Analysis of carbapenems by hydrophilic interaction chromatography and its application].

    PubMed

    Zhu, Yinfang; Ji, Shunli; Li, Shaohui; Li, Cheng; Zhang, Feifang; Liang, Xinmiao

    2015-09-01

    A hydrophilic interaction chromatographic (HILIC) method has been developed for the determination of the four carbapenems in human urine and tap water. The parameters including acetonitrile amount, buffer concentration and pH on the retention behavior of the four carbapenem antibiotics on an XAmide column were explored and the possible HILIC retention mechanism was proposed. Good linearities were obtained over the mass concentration ranges of 0.1-250 mg/L for biapenem, doripenem and ertapenem with correlation coefficients (R2) = 0.999 9 and while it was 0.5-250 mg/L with R2 = 0.999 8 for meropenem. The limits of quantification (LOQs) of all carbapenems were 0.1-0.5 mg/L. The spiked recoveries were within 100.4%-111.9% (RSD < 1%) for urine samples and 79.6%-107.4% (RSD < 5%) for tap water samples all at the spiked levels of 5 mg/L and 25 mg/L. The proposed method is accurate, sensitive, simple and suitable for the determination of the four carbapenems in human urine samples and tap water samples.

  15. Determination of Oxidized Phosphatidylcholines by Hydrophilic Interaction Liquid Chromatography Coupled to Fourier Transform Mass Spectrometry

    PubMed Central

    Sala, Pia; Pötz, Sandra; Brunner, Martina; Trötzmüller, Martin; Fauland, Alexander; Triebl, Alexander; Hartler, Jürgen; Lankmayr, Ernst; Köfeler, Harald C.

    2015-01-01

    A novel liquid chromatography-mass spectrometry (LC-MS) approach for analysis of oxidized phosphatidylcholines by an Orbitrap Fourier Transform mass spectrometer in positive electrospray ionization (ESI) coupled to hydrophilic interaction liquid chromatography (HILIC) was developed. This method depends on three selectivity criteria for separation and identification: retention time, exact mass at a resolution of 100,000 and collision induced dissociation (CID) fragment spectra in a linear ion trap. The process of chromatography development showed the best separation properties with a silica-based Kinetex column. This type of chromatography was able to separate all major lipid classes expected in mammalian samples, yielding increased sensitivity of oxidized phosphatidylcholines over reversed phase chromatography. Identification of molecular species was achieved by exact mass on intact molecular ions and CID tandem mass spectra containing characteristic fragments. Due to a lack of commercially available standards, method development was performed with copper induced oxidation products of palmitoyl-arachidonoyl-phosphatidylcholine, which resulted in a plethora of lipid species oxidized at the arachidonoyl moiety. Validation of the method was done with copper oxidized human low-density lipoprotein (LDL) prepared by ultracentrifugation. In these LDL samples we could identify 46 oxidized molecular phosphatidylcholine species out of 99 possible candidates. PMID:25874761

  16. Core shell stationary phases for a novel separation of triglycerides in plant oils by high performance liquid chromatography with electrospray-quadrupole-time of flight mass spectrometer.

    PubMed

    La Nasa, Jacopo; Ghelardi, Elisa; Degano, Ilaria; Modugno, Francesca; Colombini, Maria Perla

    2013-09-20

    A new method for the analysis of triglycerides (TAGs) in vegetable oils was developed using a Poroshell 120 EC-C18 column (3.0 mm×50 mm, 2.7 μm) with a high resolution ESI-Q-ToF tandem mass spectrometer as detection system. We used an Agilent Poroshell column, which is characterized by a recently developed stationary phase based on non-porous core particles. The results highlighted the advantages of this column in terms of the dramatic improvement in the number of theoretical plates and in low column backpressure. The developed method enabled us to analyze complex mixtures of more than 40 TAGs within less than 25 min and with a low backpressure (lower than 100 bar), and represents the first application of a core-shell stationary phase in reverse phase HPLC using an ESI-Q-ToF as detection system. The method was optimized on standards of TAGs, validated and applied to several plant oils. By a quantitative point of view, the method showed a very good linearity (r(2)>0.999) in the range 0.1-2.4 μg/g; high intra- and inter-day precision both in terms of retention times (RSD%<0.04%) and peak areas (RSD%<0.3%). Limits of detection and quantitation were lower than 0.03 μg/g and 0.10 μg/g, respectively.

  17. Preparation of a mercaptopropyl bonded silica intermediate in supercritical carbon dioxide: synthesis, characterisation and chromatography of a quinine based chiral stationary phase.

    PubMed

    Scully, Norma M; O'Sullivan, Gerard P; Healy, Liam O; Glennon, Jeremy D; Dietrich, Benjamin; Albert, Klaus

    2007-07-13

    This research examines the preparation of a mercaptopropyl bonded silica intermediate in supercritical carbon dioxide (sc-CO(2)) and the subsequent conversion in sc-CO(2) to a quinine derived chiral stationary phase (CSP). The effects of reaction temperature, pressure and time on the surface coverage of the silica intermediate were investigated when porous silica particles (Exsil-Avanti, 3microm) were reacted with 3-trimethoxymercaptopropylsilane in sc-CO(2). We present results which demonstrate that a stable mercaptopropyl bonded silica intermediate can be successfully prepared under supercritical conditions of 40 degrees C, 483bar, in a substantially reduced reaction time of 1h with superior surface coverages compared to organic solvent based methods. The further utility of this supercritical fluid technology was demonstrated by the free radical addition of a quinine derived chiral selector onto a mercaptopropyl bonded silica intermediate in sc-CO(2). This supercritical fluid generated chiral stationary phase (CSP) was utilised for the direct LC enantioseparation of a series of 3,5-dinitrobenzoyl (DNB) amino acids. Bonded silica samples were characterised using elemental analysis, diffuse reflectance infrared fourier transform (DRIFT) spectroscopy, solid state (13)C and (29)Si CP-MAS NMR spectroscopy, and thermogravimetric analysis (TGA). This supercritical fluid functionalisation approach offers an efficient and cleaner alternative to existing organic solvent based approaches for the preparation of bonded silica phases.

  18. Hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry: effect of electrospray ionization source geometry on sensitivity.

    PubMed

    Periat, Aurélie; Kohler, Isabelle; Bugey, Aurélie; Bieri, Stefan; Versace, François; Staub, Christian; Guillarme, Davy

    2014-08-22

    In this study, the influence of electrospray ionization (ESI) source design on the overall sensitivity achieved in hydrophilic interaction chromatography (HILIC) and reversed phase liquid chromatography (RPLC), was investigated. State-of-the-art triple quadrupole mass analyzers from AB Sciex, Agilent Technologies and Waters equipped with brand specific source geometries were tested with various mobile phase pH on 53 pharmaceutical compounds. The design of the ESI source showed to strongly influence the gain in sensitivity that can be achieved in HILIC compared to RPLC mode. The 6460 Triple Quadrupole LC/MS system from Agilent Technologies was particularly affected by mobile phase settings. Indeed, compared to RPLC conditions, 92% of the compounds had an increased signal-to-noise ratio at a flow rate of 300 μL/min in HILIC mode at pH 6, while this percentage dropped to only 7% at 1000 μL/min and pH 3. In contrast, the influence of flow rate and mobile phase pH on the gain in sensitivity between RPLC and HILIC was found very limited with the API 5000 LC/MS/MS system from AB Sciex, as only 15 to 36% of the tested compounds showed an enhanced sensitivity in HILIC mode. With the Xevo TQ-S instrument from Waters, superior sensitivity in HILIC was noticed for 85% of the compounds with optimal conditions (i.e., pH 3 and 1000 μL/min), whereas at sub-optimal conditions (i.e. pH 6 and 300 μL/min), it represented less than 50%. The gain in sensitivity observed in HILIC was found less significant with the recent LC-MS platforms used in this study than for old-generation instruments. Indeed, the improved ESI sources equipping the recent mass analyzers allow for enhanced evaporation efficiency, mainly for RPLC mobile phases containing high proportion of water and this even at high flow rates.

  19. Hydrophilic interaction liquid chromatography tandem mass spectrometry analysis of malonyl-coenzyme A in breast cancer cell cultures applying online solid-phase extraction.

    PubMed

    Schriewer, Alexander; Cadenas, Cristina; Hayen, Heiko

    2017-09-06

    Cofactors such as coenzyme A and its derivatives acetyl-coenzyme A and malonyl-coenzyme A are involved in many metabolic pathways. Due to trace level concentrations in biological samples and the high reactivity of cofactors, a fast, sensitive and selective method for quantification is mandatory. In this study, online solid-phase extraction was coupled successfully to hydrophilic interaction liquid chromatography with tandem mass spectrometry for analytes' isolation in complex matrix and quantification by external calibration. Online solid-phase extraction was carried out by application of a weak anion exchange column, whereas hydrophilic interaction liquid chromatography separation was performed on an amide modified stationary phase. Sample preparation of the extracts prior the analysis was reduced to a centrifugation and dilution step. Moreover, the applied online solid-phase extraction significantly reduced matrix effects and increased the signal-to-noise ratio. The limit of detection and the limit of quantification were in the lower nanomolar range. Finally, the applicability of this method was demonstrated on MCF-7 breast cancer cell cultures, a common used model system, where acetyl-coenzyme A and malonyl-coenzyme A were determined with standard addition procedure in concentrations of 1.98 μM and 41 nM, respectively. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. TLC-MS versus TLC-LC-MS fingerprints of herbal extracts. Part III. Application of the reversed-phase liquid chromatography systems with C18 stationary phase.

    PubMed

    Sajewicz, Mieczysław; Staszek, Dorota; Natic, Maja; Waksmundzka-Hajnos, Monika; Kowalska, Teresa

    2011-08-01

    In the previous paper from this series, we proposed mass spectrometric fingerprinting of complex botanical samples upon the examples of the pharmacologically important phenolic acids and flavonoids selectively extracted from Salvia lavandulifolia. In this study, we explore fingerprinting efficiency with a novel two-dimensional analytical system composed of the reversed-phase thin-layer chromatography and the reversed-phase high performance liquid chromatography with mass spectrometric detection (2D RP-TLC-RP-LC-MS). We also compare its efficiency with that of the one-dimensional analytical system (the reversed-phase thin-layer chromatography with mass spectrometric detection; 1D RP-TLC-MS). As our present study is basically focused on the method development, we considered it as justified to carry out our comparison with the phenolic acid extracts selectively derived from the Salvia lavandulifolia species, similar as it was done in Part II from this series. Upon the results obtained, it was established that the 1D RP-TLC-MS mode and the 2D RP-TLC-RP-LC-MS mode can be applied to fingerprinting of herbal extracts, and that the 2D RP-TLC-RP-LC mode can provide a more abundant information than that originating from the 1D RP-TLC mode.

  1. Synthesis of sugar-based silica gels by copper-catalysed azide-alkyne cycloaddition via a single-step azido-activated silica intermediate and the use of the gels in hydrophilic interaction chromatography.

    PubMed

    Moni, Lisa; Ciogli, Alessia; D'Acquarica, Ilaria; Dondoni, Alessandro; Gasparrini, Francesco; Marra, Alberto

    2010-05-17

    Novel sugar-based silica gels were prepared by exploiting the copper-catalysed azide-alkyne cycloaddition (CuAAC) of two different sugar alkynes, namely, ethynyl C-galactoside 1 and propargyl O-lactoside 2, with new single-step azido-activated silica gels. The fully characterised stationary phases were generally used for hydrophilic interaction chromatography (HILIC), with particular application in the stereoselective separation of monosaccharides. Dynamic HILIC (DHILIC) experiments were performed to evaluate the influence of mutarotation on the chromatographic peak shapes of two interconverting sugar anomers. The potential of such materials was shown in the separation of other highly polar compounds, including amino acids and flavonoids.

  2. Analysis of hydrophilic metabolites by high-performance liquid chromatography-mass spectrometry using a silica hydride-based stationary phase.

    PubMed

    Pesek, Joseph J; Matyska, Maria T; Fischer, Steven M; Sana, Theodore R

    2008-09-12

    A novel silica hydride-based stationary phase was used to evaluate the retention behavior in the aqueous normal-phase (ANP) mode of standards representing three classes of metabolites. The effects on retention behavior of amino acids, carbohydrates and small organic acids were examined by altering the column temperature, and by adding different additives to both the mobile phase and sample solvent. Gradient mode results revealed the repeatability of retention times to be very stable for these compound classes. At both 15 and 30 degrees C, excellent RSD values were obtained with less than 1% variation for over 50 injections of an amino acid mixture. The ability to separate the 19 nonderivatized amino acid standards, organic acids and carbohydrates was demonstrated as well as the potential for this material to separate polar metabolites in complex fluids such as urine.

  3. Preparation of poly(glycidylmethacrylate-divinylbenzene) weak acid cation exchange stationary phases with succinic anhydride, phthalic anhydride, and maleic anhydride for ion chromatography.

    PubMed

    Liu, Junwei; Wang, Yong; Wu, Shuchao; Zhang, Peimin; Zhu, Yan

    2016-08-01

    In this work, poly(glycidylmethacrylate-divinylbenzene) microspheres were prepared and applied for the preparation of weak acid cation exchange stationary phases. Succinic anhydride, phthalic anhydride, and maleic anhydride were selected as carboxylation reagents to prepare three weak acid cation exchangers by direct chemical derivatization reaction without solvent or catalyst. The diameters and dispersity of the microspheres were characterized by scanning electron microscopy; the amount of accessible epoxy groups and mechanical stability were also measured. The weak acid cation exchangers were characterized by Fourier transform infrared spectroscopy; the content of carboxyl groups was measured by traditional acid base titration method. The chromatographic properties were characterized and compared by separating alkali, alkaline earth metal ions and ammonium and polar amines. The separation properties enhanced in the order of succinic anhydride, phthalic anhydride, and maleic anhydride modified poly(glycidylmethacrylate-divinylbenzene) cation exchangers.

  4. Characterisation of reversed-phase liquid chromatography stationary phases for the analysis of basic pharmaceuticals: eluent properties and comparison of empirical test methods.

    PubMed

    Vervoort, R J; Ruyter, E; Debets, A J; Claessens, H A; Cramers, C A; de Jong, G J

    2001-10-05

    The reversed-phase liquid chromatographic analysis of basic pharmaceuticals can be problematic. Both the properties of the eluent and the stationary phase can influence the chromatographic performance. Therefore selection of suitable experimental conditions for the analysis of basic compounds can be difficult. This paper shows that the organic modifier and the nature of the buffer influence the eluent properties. Moreover, the nature and amount of modifier also influence the basicity of the analytes. Investigations showed that the nature of the buffer can have a significant influence on retention and peak shape of basic compounds. Test procedures using basic analytes as test probes provided relevant information with respect to selecting columns to analyse basic pharmaceutical compounds. Test procedures using compounds like aniline, phenol and benzene were found to be less suitable.

  5. Investigation on enantiomeric separations of fluorenylmethoxycarbonyl amino acids and peptides by high-performance liquid chromatography using native cyclodextrins as chiral stationary phases.

    PubMed

    Tang, Y; Zukowski, J; Armstrong, D W

    1996-09-06

    A systematic study was carried out to investigate enantiomeric separations of fluorenylmethoxycarbonyl (FMOC) amino acids and their peptides. Twenty amino acids were derivatized by 9-fluorenylmethyl chloroformate (FMOC-Cl) and its analogues, FMOC-glycyl-Cl and FMOC-beta-alanyl-Cl. All derivatives were chromatographed on native beta- and gamma-cyclodextrin columns using acetonitrile as the main mobile phase component. The results indicated that glycyl and beta-alanyl groups between FMOC and amino acid moieties enhanced chiral selectivities of amino acid derivatives. The addition of modifiers, triethylamine, acetic acid and methanol, into the mobile phase caused alterations in retention, enantiorecognition and elution order. The structures of amino acids and the type of chiral stationary phase employed exhibited significant impacts on chiral resolutions. It is also found that the number and position of glycyl moieties affect the retentions and enantioselectivities of FMOC derivatized glycyl containing peptides.

  6. Comparison of various types of stationary phases in non-aqueous reversed-phase high-performance liquid chromatography-mass spectrometry of glycerolipids in blackcurrant oil and its enzymatic hydrolysis mixture.

    PubMed

    Lísa, Miroslav; Holcapek, Michal; Sovová, Helena

    2009-11-20

    The selection of column packing during the development of high-performance liquid chromatography method is a crucial step to achieve sufficient chromatographic resolution of analyzed species in complex mixtures. Various stationary phases are tested in this paper for the analysis of complex mixture of triacylglycerols (TGs) in blackcurrant oil using non-aqueous reversed-phase (NARP) system with acetonitrile-2-propanol mobile phase. Conventional C(18) column in the total length of 45 cm is used for the separation of TGs according to their equivalent carbon number, the number and positions of double bonds and acyl chain lengths. The separation of TGs and their more polar hydrolysis products after the partial enzymatic hydrolysis of blackcurrant oil in one chromatographic run is achieved using conventional C(18) column. Retention times of TGs are reduced almost 10 times without the loss of the chromatographic resolution using ultra high-performance liquid chromatography with 1.7 microm C(18) particles. The separation in NARP system on C(30) column shows an unusual phenomenon, because the retention order of TGs changes depending on the column temperature, which is reported for the first time. The commercial monolithic column modified with C(18) is used for the fast analysis of TGs to increase the sample throughput but at cost of low resolution.

  7. Protein losses in ion-exchange and hydrophobic interaction high-performance liquid chromatography

    SciTech Connect

    Goheen, Steven C.; Gibbins, Betty M.

    2000-01-01

    Protein losses in ion-exchange and hydrophobic interaction HPLC were examined. The supports were allnon-porous, packed in columns of identical dimensions. Two ion-exchange chromatography (IEC), anion and cation, as well as a hydrophobic interaction chromatography (HIC) columns were tested. Proteins included cytochrome c, bovine serum albumin (BSA), immunoglobulin G and fibrinogen. Temperature effects on HIC supports were studied for cytochrome c and BSA. Both retention times and recoveries of the proteins were measured. The influence of column residence time on the recovery of proteins were also investigated. We found a linear relationship between the amount of protein recovered and the log of the molecular mass. Retention times also generally increased with temperature for both HIC and IEC. Other trends in retention behavior and recoveries are discussed.

  8. Dynamic high performance liquid chromatography on chiral stationary phases. Low temperature separation of the interconverting enantiomers of diazepam, flunitrazepam, prazepam and tetrazepam.

    PubMed

    Sabia, Rocchina; Ciogli, Alessia; Pierini, Marco; Gasparrini, Francesco; Villani, Claudio

    2014-10-10

    Diazepam and the structurally related 1,4-benzodiazepin-2-ones tetrazepam, prazepam and flunitrazepam are chiral molecules because they adopt a ground state conformation featuring a non-planar seven membered ring devoid of any reflection-symmetry element. The two conformational enantiomers of this class of benzodiazepines interconvert rapidly at room temperature by a simple ring flipping process. Low temperature HPLC on the Whelk-O1 chiral stationary phase allowed us to separate the conformational enantiomers of diazepam and of the related 1,4-benzodiazepin-2-ones, under conditions where the interconversion rate is sufficiently low, compared to the chromatographic separation rate. Diazepam, tetrazepam and prazepam showed temperature dependent dynamic HPLC profiles with interconversion plateaus indicative of on-column enantiomer interconversion (enantiomerization) in the temperature range between -10 °C and -35 °C, whereas for flunitrazepam on-column interconversion was observed at temperatures between -40 °C and -66 °C. Simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At -20 °C the enantiomerization barriers, ΔG(≠), for diazepam, prazepam and tetrazepam were determined to be in the range 17.6-18.7 kcal/mol. At -55 °C ΔG(≠) for flunitrazepam was determined to be in the 15.6-15.7 kcal/mol range. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper call for a reinterpretation of previously published results on the HPLC behavior of diazepam on chiral stationary phases. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Self-interaction chromatography in pre-packed columns: a critical evaluation of self-interaction chromatography methodology to determine the second virial coefficient.

    PubMed

    Rakel, Natalie; Schleining, Kristina; Dismer, Florian; Hubbuch, Juergen

    2013-06-07

    The characterization of protein-protein interactions is commonly conducted via self-interaction chromatography to describe magnitude and direction of the interactions with the resulting osmotic second virial coefficient (B22). However, the method is invasive and protein immobilization on the adsorber surface can influence the results obtained. In order to replace batch immobilization procedures followed by a column packing, direct on-column preparation was optimized in terms of protein immobilization under a continuous flow. Surface load was measured applying a novel method based on partial least squares analysis of spectral scans to reduce analytical error when determining the amount of immobilized protein. Subsequently influencing parameters such as the effects of absolute surface load, injected protein concentration and distribution of protein orientation were analyzed and system performance evaluated. The results disprove the consistency of the SIC method regarding the non-random orientation of proteins on adsorber particles. Thus the determined B22-values differ quantitatively from those determined with static light scattering. Furthermore, variations in immobilization conditions influence the results obtained. These results make clear that SIC does not fulfill the theoretical framework of B22-analysis. It is rather a qualitative measure of protein-protein interactions in the respective system used for experimentation.

  10. Hydrophilic interaction chromatography with aerosol-based detectors (ELSD, CAD, NQAD) for polar compounds lacking a UV chromophore in an intravenous formulation.

    PubMed

    Cintrón, José M; Risley, Donald S

    2013-05-05

    In this work, a high performance liquid chromatography (HPLC) method is reported for the separation and quantitation of a drug substance that is highly polar and lacking a chromophore in a mannitol intravenous (IV) formulation. Three polar stationary phases operated in hydrophilic interaction chromatography (HILIC) mode were evaluated in conjunction with an Alltech 800 ELSD detector. These columns were evaluated with respect to chromatographic properties such as buffer, pH and organic concentrations to identify the best stationary phase. The chromatographic method was then validated for the determination of mGlu2/3 receptor agonist (-)-(1R, 4S, 5S, 6S)-4-Amino-2-sulfonylbicyclo [3.1.0] hexane-4,6-dicarboxylic acid (LY404039) content in a mannitol IV formulation with respect to linearity (R(2) of 0.9997), repeatability (%RSD of 0.36%), accuracy, solution stability (99.56% after 24h), specificity, intra-assay precision (%RSD 0.48%) and limit of detection (LOD, ∼50 μg/mL). In addition to the Alltech 800 ELSD detector, several other aerosol-based detectors were investigated for reproducibility, linearity and LOD. These additional detectors consisted of an Alltech 3300 evaporative light scattering detector (ELSD), a nano quantity analyte detector (NQAD) and a charged aerosol detector (CAD). Based on the data from this report, a feasible isocratic LC method was achieved using a TSKgel Amide-80 column with mobile phase conditions of 30% water with 0.2% trifluoroacetic acid (TFA) and 70% acetonitrile (ACN) using any of the four aerosol-based detectors for detection and quantitation.

  11. C₁₈-bound porous silica monolith particles as a low-cost high-performance liquid chromatography stationary phase with an excellent chromatographic performance.

    PubMed

    Ali, Faiz; Cheong, Won Jo

    2014-12-01

    Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol-gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end-capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185,000/m) and chromatographic resolution were achieved when the C18 -bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless-steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60,000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C18 modification in addition to partial-monolithic architecture has resulted in a useful stationary phase (C18 -bound silica monolith particles) of low production cost showing excellent chromatographic performance.

  12. The enantioselective determination of chlorpheniramine and its major metabolites in human plasma using chiral chromatography on a beta-cyclodextrin chiral stationary phase and mass spectrometric detection.

    PubMed

    Fried, Karen M; Young, Andrea E; Usdin Yasuda, Sally; Wainer, Irving W

    2002-01-15

    A sensitive enantioselective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of plasma concentrations of (-)(R)- and (+)(S)-chlorpheniramine (CP) and their metabolites, desmethyl-chlorpheniramine (DCP), didesmethyl-chorpheniramine (DDCP) and chlorpheniramine N-oxide (CPNO). Enantioselective separations were achieved on a beta-cyclodextrin chiral stationary phase (CYCLOBOND I 2000) with a mobile phase consisting of diethylamine acetate (0.25%, pH 4.4):methanol:acetonitrile [85:7.5:7.5, (v/v/v)]and a flow-rate of 0.5 ml/min. For CP, the enantioselectivity (alpha) of the separation was 1.12 with a resolution factor (R(s)) of 1.17. The method was validated for CP by using mass spectroscopy detection (MSD). Concentrations of each enantiomer could be measured down to 125 pg/ml from a 1-ml plasma sample. Extracted calibration curves were linear from 0.13 to 50.00 ng/ml for each enantiomer. The method was applied to samples from two clinical studies.

  13. Enantiomeric separation of volatile organics by gas chromatography for the in situ analysis of extraterrestrial materials: kinetics and thermodynamics investigation of various chiral stationary phases.

    PubMed

    Freissinet, C; Buch, A; Szopa, C; Sternberg, R

    2013-09-06

    The performances of several commercial chiral capillary columns have been evaluated with the aim of determining the one most suitable for enantiomeric separation in a gas chromatograph onboard a space probe. We compared the GC-MS response of three capillary columns coated with different chiral stationary phases (CSP) using volatile chiral organic molecules which are potential markers of a prebiotic organic chemistry. The three different chiral capillary columns are Chirasil-Val, with an amino acid derivative CSP, ChiralDex-β-PM, with a CSP composed of dissolved permethylated β-cyclodextrins in polysiloxane, and Chirasil-Dex, with a CSP made of modified cyclodextrins chemically bonded to the polysiloxane backbone. Both kinetics and thermodynamics studies have been carried out to evaluate the chiral recognition potential in these different types of columns. The thermodynamic parameters also allow a better understanding of the driving forces affecting the retention and separation of the enantiomers. The Chirasil-Dex-CSP displays the best characteristics for an optimal resolution of the chiral compounds, without preliminary derivatization. This CSP had been chosen to be the only chiral column in the Sample Analysis at Mars (SAM) experiment onboard the current Mars Science Laboratory (MSL) mission, and is also part of the Mars Organic Molecules Analyzer (MOMA) gas chromatograph onboard the next Martian mission ExoMars. The use of this column could also be extended to all space missions aimed at studying chirality in space.

  14. [Comparative enantiomer separation of beta-blockers on polysaccharide derived chiral stationary phases using high performance liquid chromatography with acid or base additive in the mobile phases].

    PubMed

    Huang, Hu; Jin, Jingyu; Lee, Wonjae

    2009-07-01

    The liquid chromatographic enantiomer separation of beta-blockers on several polysaccharide derived chiral stationary phases (CSPs) (Chiralpak AD, Chiralcel OD, Chiralpak IA and Chiralpak IB) was performed and compared in the normal phase mode using hexane-ethanol in the presence of acid or base additives. The chromatographic conditions were 10% - 30% (v/v) ethanol-hexane containing 0.1% trifluoroacetic acid or triethylamine as the mobile phase at the flow rate of 1.0 mL/min with the detection at 254 nm. The lower enantioselectivities and the shorter retention times on amylose derived CSPs (Chiralpak AD and Chiralpak IA) using the mobile phase with acid additive than those with base additive were shown, except for slightly longer retention times of metoprolol and propranolol on Chiralpak AD. The greater enantioselectivities and the shorter retention times on cellulose derived CSPs (Chiralcel OD and Chiralpak IB) using the mobile phase with acid additive than those with base additive were shown, especially, Chiralcel OD showed dramatically enhanced enantioselectivites using the mobile phase with acidic additive. Also, it was shown that the greater enantiomer separation of beta-blockers on Chiralcel OD was achieved using the mobile phases with the higher concentration acid additive.

  15. Analytical and semipreparative high performance liquid chromatography enantioseparation of bicalutamide and its chiral impurities on an immobilized polysaccharide-based chiral stationary phase.

    PubMed

    Sadutto, Daniele; Ferretti, Rosella; Zanitti, Leo; Casulli, Adriano; Cirilli, Roberto

    2016-05-06

    Direct HPLC separation of enantiomers of Bicalutamide (BCT), a non-steroidal antiandrogen used for the treatment of prostate cancer, was performed by using the immobilized amylose-based Chiralpak IA chiral stationary phase (CSP). Enantioselective conditions were achieved using standard normal phase mixtures n-hexane-alcohol (ethanol or 2-propanol) and a "non-standard" mobile phase containing ethyl acetate (EA). The chromatographic behaviour of the IA CSP under these elution modes was evaluated and compared at different temperatures. The eluent mixture n-hexane-EA-ethanol 100-30-5 (v/v/v) and the column temperature of 40°C were identified as the best operational conditions to carry out semipreparative enantioseparations on a 1-cm I.D. IA column. Using this protocol, about 960mg of (R)-BCT, which is the enantiomer with the almost entire anti-androgenic activity of BCT, per day could be isolated. The analytical and semipreparative HPLC resolution of chiral impurities of BCT, and their empiric absolute configuration assignment by circular dichroism correlation method are also presented.

  16. Analysis of benidipine enantiomers in human plasma by liquid chromatography-mass spectrometry using a macrocyclic antibiotic (Vancomycin) chiral stationary phase column.

    PubMed

    Kang, Wonku; Lee, Dong-Jun; Liu, Kwang-Hyeon; Sunwoo, Yu Eun; Kwon, Kwang-il; Cha, In-June; Shin, Jae-Gook

    2005-01-05

    We used a novel chromatographic method to rapidly and simply characterize the pharmacokinetics of benidipine enantiomers in human plasma. The stereoisomers of benidipine were extracted from plasma using diethylether under alkaline conditions. After evaporating the organic layer, the residue was reconstituted in the mobile phase (methanol:acetic acid:triethylamine, 100:0.01:0.0001, v/v/v). The enantiomers in the extract were separated on a macrocyclic antibiotic (Vancomycin) chiral stationary phase column. The mobile phase was eluted at 1 ml/min and was split by an interface. One-fifth of the eluent was used to quantify both isomers in a tandem mass spectrometer in multiple reaction-monitoring mode. The coefficient of variation of the precision of the assay was less than 8%, the assay accuracy was between 93.4 and 113.3%, and the limit of detection was 0.05 ng/ml for 1 ml of plasma. The method described above was used to measure the concentration of both benidipine enantiomers in plasma from healthy subjects who received a single oral dose of a racemate of 8 mg benidipine. The C(max) and AUC(inf) values of (+)-alpha benidipine were higher than those of (-)-alpha benidipine by 1.96- and 1.85-fold, respectively (p<0.001), whereas, the T(max) and t(1/2) for each of the benidipine stereoisomers were not significantly different.

  17. Combination of two different stationary phases for on-line pre-concentration and separation of basic drugs by using nano-liquid chromatography.

    PubMed

    D'Orazio, Giovanni; Fanali, Salvatore

    2013-04-12

    Capillary columns were packed firstly with silica modified-teicoplanin (teico-CSP) particles for a short zone (1-5 cm) and then with a Cogent Bidentate C18 silica phase (25 cm). The first part of the column (inlet) was intended for focusing the sample model, consisted of selected basic compounds, while the second zone, containing RP18 particles, was used for their separation. For method optimization, some important experimental parameters were studied including the sample solvent, injected volume and teico-CSP particles length. 3 cm teico-CSP resulted to be effective for the on-line pre-concentration, before the separation, of acebutolol, alprenolol, nadolol, oxprenolol and terbutaline with limit of detection at levels of few ng/mL. The comparison of the data obtained in absence of the chiral stationary phase revealed that the use of this chiral short sector into the capillary allowed the increase of the sensitivity of 5-12 times. Injection of larger sample volumes were easily done using higher length of the teico-CSP into the capillary, however the use of 5 cm length was not appropriate because caused the partial chiral separation of some studied compounds.

  18. Screening for low molecular weight compounds in fish meal solubles by hydrophilic interaction liquid chromatography coupled to mass spectrometry

    USDA-ARS?s Scientific Manuscript database

    A simple analytical method using hydrophilic interaction liquid chromatography coupled with mass spectrometry was developed to screen for low molecular weight compounds in enzyme treated and untreated Alaskan pollock (Theragra chalcogramma) stickwater (SW) generated from processing fish meal with po...

  19. Comparison of zonal elution and nonlinear chromatography in determination of the interaction between seven drugs and immobilised β(2)-adrenoceptor.

    PubMed

    Li, Qian; Wang, Jing; Zheng, Yuqing Yuan; Yang, Lingjian; Zhang, Yajun; Bian, Liujiao; Zheng, Jianbin; Li, Zijian; Zhao, Xinfeng; Zhang, Youyi

    2015-07-03

    Zonal elution and nonlinear chromatography are two mainstream models for the determination of drug-protein interaction in affinity chromatography. This work intended to compare the results by zonal elution with that by nonlinear chromatography when it comes to the analysis of the interaction between seven drugs and immobilised β2-adrenoceptor (β2-AR). The results of the zonal elution showed that clorprenaline, clenbuterol, methoxyphenamine, salbutamol, terbutaline, tulobuterol and bambuterol have only one type of binding site on immobilised β2-AR, while nonlinear chromatography confirmed the existence of at least two types of binding sites between β2-AR and clorprenaline, clenbuterol and bambuterol. On these sites, both zonal elution and nonlinear chromatography presented the same rank order for the association constants of the seven drugs. Compared with the data from zonal elution, the association constants calculated using nonlinear chromatography gave a good linear response to the corresponding values by radio-ligand binding assay. The sampling efficiencies of nonlinear chromatography were clearly higher than zonal elution. Nonlinear chromatography will probably become a powerful alternative for the high throughput determination of drug-protein interaction.

  20. Purification of tracer for somatomedin C radioimmunoassay by hydrophobic interaction chromatography

    SciTech Connect

    Baxter, R.C.; Brown, A.S.

    1982-03-01

    A tracer for use in the somatomedin C radiommunoassay by hydrophobic interaction chromatography was purified. Material showing greatest immunoreactivity binds to Octyl Sepharose CL-4B (Pharmacia) in a buffer mixture consisting of 130 mL of acetonitrile and 870 mL of 0.1 mol/L NH/sub 4/HCO/sub 3/, pH 7.8, but is eluted by increasing the acetonitrile content to 180 mL/L. As compared with tracer purified by binding to specific antiserum in liquid phase, precipitating the complex with second antibody, and then dissociating by gel chromatography at acid pH, this tracer shows equal immunoreactivity against specific somatomedin C antiserum. Either preparation allows excellent discrimination between extracts of normal, acromegalic, and hypopituitary plasma samples; thus either is suitable for use in the somatomedin C radioimmunoassay. Tracer purification by hydrophobic interaction chromatography is rapid and inexpensive. It may be useful in preparing highly immunoreactive tracers for other peptide radioimmunoassays.

  1. Evaluation of a hydrophilic interaction liquid chromatography design space for sugars and sugar alcohols.

    PubMed

    Hetrick, Evan M; Kramer, Timothy T; Risley, Donald S

    2017-03-17

    Based on a column-screening exercise, a column ranking system was developed for sample mixtures containing any combination of 26 sugar and sugar alcohol analytes using 16 polar stationary phases in the HILIC mode with acetonitrile/water or acetone/water mobile phases. Each analyte was evaluated on the HILIC columns with gradient elution and the subsequent chromatography data was compiled into a statistical software package where any subset of the analytes can be selected and the columns are then ranked by the greatest separation. Since these analytes lack chromophores, aerosol-based detectors, including an evaporative light scattering detector (ELSD) and a charged aerosol detector (CAD) were employed for qualitative and quantitative detection. Example qualitative applications are provided to illustrate the practicality and efficiency of this HILIC column ranking. Furthermore, the design-space approach was used as a starting point for a quantitative method for the trace analysis of glucose in trehalose samples in a complex matrix. Knowledge gained from evaluating the design-space led to rapid development of a capable method as demonstrated through validation of the following parameters: specificity, accuracy, precision, linearity, limit of quantitation, limit of detection, and range.

  2. Stationary phase thickness determines the quality of thin-layer chromatography/matrix-assisted laser desorption and ionization mass spectra of lipids.

    PubMed

    Griesinger, Hans; Fuchs, Beate; Süß, Rosmarie; Matheis, Katerina; Schulz, Michael; Schiller, Jürgen

    2014-04-15

    Normal phase thin-layer chromatography (NP TLC) is an established method of (phospho)lipid analysis. The determination of the fatty acyl composition is, however, a more challenging task by NP TLC. The direct coupling of TLC separation with mass spectrometric detection (e.g., matrix-assisted laser desorption/ionization mass spectrometry, MALDI MS), however, enables a detailed characterization of complex lipid mixtures. Here we show that the thickness of the silica gel layer has a considerable effect on the quality of the mass spectra recorded directly from the TLC plate. In particular, the intensity of the matrix background signals can be reduced if "thinner" TLC layers are used. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Methacrylate-based diol monolithic stationary phase for the separation of polar and non-polar compounds in capillary liquid chromatography.

    PubMed

    Linda, Roza; Lim, Lee Wah; Takeuchi, Toyohide

    2013-01-01

    A monolithic capillary column prepared with glycidyl methacrylate (GMA) and poly(ethylene glycol) dimethacrylate (PEGDMA) was investigated and used in capillary liquid chromatography. The polymer monolith was synthesized in the presence of methanol and decanol as the biporogenic solvents by in situ polymerization of GMA and PEGDMA, and the optimum composition of monomer and porogen was investigated. After polymerization, glycidyl groups were hydrolyzed with sulfuric acid to produce diol groups at the surface of the porous monolith via epoxy-ring-opening. The GMA content in the polymerization mixture affected the hydrophilicity of the monolith. The separation capability was evaluated by separation of phenol compounds, phthalic acids, and polycyclic aromatic hydrocarbons. The poly(GMA-PEGDMA) monolithic capillary column exhibited satisfactory stability.

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

    PubMed

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

    2014-01-01

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

  5. Off-line comprehensive 2-dimensional hydrophilic interaction x reversed phase liquid chromatography analysis of procyanidins.

    PubMed

    Kalili, Kathithileni M; de Villiers, André

    2009-08-28

    The development of an off-line comprehensive 2-dimensional liquid chromatography (2-D-LC) method for the analysis of procyanidins is reported. In the first dimension, oligomeric procyanidins were separated according to molecular weight by hydrophilic interaction chromatography (HILIC), while reversed phase LC was employed in the second dimension to separate oligomers based on hydrophobicity. Fluorescence, UV and electrospray ionisation mass spectrometry (ESI-MS) were employed for identification purposes. The combination of these orthogonal separation methods is shown to represent a significant improvement compared to 1-dimensional methods for the analysis of complex high molecular weight procyanidin fractions, by simultaneously providing isomeric and molecular weight information. The low correlation (r(2)<0.2100) between the two LC modes afforded a practical peak capacity in excess of 2300 for the optimal off-line method. The applicability of the method is demonstrated for the analysis of phenolic extracts of apple and cocoa.

  6. Studying host cell protein interactions with monoclonal antibodies using high throughput protein A chromatography.

    PubMed

    Sisodiya, Vikram N; Lequieu, Joshua; Rodriguez, Maricel; McDonald, Paul; Lazzareschi, Kathlyn P

    2012-10-01

    Protein A chromatography is typically used as the initial capture step in the purification of monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Although exploiting an affinity interaction for purification, the level of host cell proteins in the protein A eluent varies significantly with different feedstocks. Using a batch binding chromatography method, we performed a controlled study to assess host cell protein clearance across both MabSelect Sure and Prosep vA resins. We individually spiked 21 purified antibodies into null cell culture fluid generated with a non-producing cell line, creating mock cell culture fluids for each antibody with an identical composition of host cell proteins and antibody concentration. We demonstrated that antibody-host cell protein interactions are primarily responsible for the variable levels of host cell proteins in the protein A eluent for both resins when antibody is present. Using the additives guanidine HCl and sodium chloride, we demonstrated that antibody-host cell protein interactions may be disrupted, reducing the level of host cell proteins present after purification on both resins. The reduction in the level of host cell proteins differed between antibodies suggesting that the interaction likely varies between individual antibodies but encompasses both an electrostatic and hydrophobic component.

  7. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.

    PubMed

    McNulty, Dean E; Annan, Roland S

    2008-05-01

    The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.

  8. Stationary phase deposition based on onium salts

    DOEpatents

    Wheeler, David R.; Lewis, Patrick R.; Dirk, Shawn M.; Trudell, Daniel E.

    2008-01-01

    Onium salt chemistry can be used to deposit very uniform thickness stationary phases on the wall of a gas chromatography column. In particular, the stationary phase can be bonded to non-silicon based columns, especially microfabricated metal columns. Non-silicon microfabricated columns may be manufactured and processed at a fraction of the cost of silicon-based columns. In addition, the method can be used to phase-coat conventional capillary columns or silicon-based microfabricated columns.

  9. Separation of monosaccharides by hydrophilic interaction chromatography with evaporative light scattering detection.

    PubMed

    Karlsson, Göran; Winge, Stefan; Sandberg, Helena

    2005-10-28

    Hydrophilic interaction liquid chromatography (HILIC) was used to separate monosaccharides that are common in N-linked oligosaccharides in glycoproteins and other compounds. A TSKgel Amide-80 column was eluted with 82% acetonitrile, in 5 mM ammonium formate (pH 5.5). Column temperature was 60 degrees C and evaporative light scattering was used for detection (ELSD). With this method, L-fucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetylneuraminic acid, and D-glucuronic acid were separated, with detection limits of 0.3-0.5 microg for each monosaccharide, and intermediate precisions were 3-6% RSD (n=6).

  10. Determination of histamine in seafood by hydrophilic interaction chromatography/tandem mass spectrometry.

    PubMed

    Yoshida, Tatsuo; Hamada, Hirotoshi; Murakawa, Hiroshi; Yoshimoto, Hidekazu; Tobino, Toshiaki; Toda, Kei

    2012-01-01

    A simple method was developed to determine histamine, an important compound in chemical food poisoning, by extraction followed by hydrophilic interaction chromatography-tandem mass spectrometry using a hydrophilic column with sulfobetaine-type zwitterion groups. The quantitation range in seafood products was from 0.4 to 200 mg kg(-1) for 5 g food samples. Quantitative recoveries were obtained with four types of seafood product. These results agreed well with those from the more complex, conventional HPLC method, which requires sample derivatization with dansyl chloride.

  11. Heparin stability by determining unsubstituted amino groups using hydrophilic interaction chromatography mass spectrometry.

    PubMed

    Fu, Li; Li, Lingyun; Cai, Chao; Li, Guoyun; Zhang, Fuming; Linhardt, Robert J

    2014-09-15

    The thermal instability of the anticoagulant heparin is associated, in part, with the solvolytic loss of N-sulfo groups. This study describes a new method to assess the increased content of unsubstituted amino groups present in thermally stressed and autoclave-sterilized heparin formulations. N-Acetylation of heparin samples with acetic anhydride-d6 is followed by exhaustive heparinase treatment and disaccharide analysis by hydrophilic interaction chromatography mass spectrometry (HILIC-MS). The introduction of a stable isotopic label provides a sensitive probe for the detection and localization of the lost N-sulfo groups, potentially providing valuable insights into the degradation mechanism and the reasons for anticoagulant potency loss.

  12. Evaluation of the dual retention properties of stationary phases based on silica hydride: Perfluorinated bonded material.

    PubMed

    Pesek, Joseph J; Matyska, Maria T; Natekar, Harshada

    2016-03-01

    The synthesis of a new perfluorinated stationary phase based on silica hydride using a hydrosilation reaction was investigated. The material was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and (13) C cross-polarization magic-angle spinning NMR spectroscopy. The retention properties of this new material were tested in the reversed-phase and normal-phase mode. Variable buffer strength experiments at two pH conditions for selected polar compounds were used to compare the new phase to hydrophilic interaction liquid chromatography retention. These results and previous data reported in the literature were used to postulate differences in the retention mechanism between hydrophilic interaction liquid chromatography and silica hydride-based stationary phases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Similar interaction chromatography of proteins: A cross interaction chromatographic approach to estimate the osmotic second virial coefficient.

    PubMed

    Quigley, A; Williams, D R

    2016-08-12

    Self-interaction chromatography (SIC) has established itself as an important experimental technique for the measurement of the second osmotic virial coefficients B22. B22 data are critical for understanding a range of protein solution phenomena, particularly aggregation and crystallisation. A key limitation to the more extensive use of SIC is the need to develop a method for immobilising each specific protein of interest onto a chromatographic support. This requirement is both a time and protein consuming constraint, which means that SIC cannot be used as a high throughput method for screening a wide range of proteins and their variants. Here an experimental framework is presented for estimating B22 values using Similar Interaction Chromatography (SimIC). This work uses experimental B23 and B32 data for lysozyme, lactoferrin, catalase and concanavalin A to reliably estimate B22 using arithmetic mean field approximations and is demonstrated to give good agreement with SIC measurements of B22 for the same proteins. SimIC could form the basis of a rapid protein variant screening methods to assess the developability of protein therapeutic candidates for industrial and academic researchers with respect to aggregation behaviour by eluting target proteins through a series of well-characterised protein immobilized reference columns. Copyright © 2016. Published by Elsevier B.V.

  14. [[Chiral separation of five arylpropionic acid drugs and determination of their enantiomers in pharmaceutical preparations by reversed-phase high performance liquid chromatography with cellulose-tris-(4-methylbenzoate) stationary phase

    PubMed

    Luo, An; Wan, Qiang; Fan, Huajun; Chen, Zhi; Wu, Xuehao; Huang, Xiaowen; Zang, Linquan

    2014-09-01

    Chromatographic behaviors for enantiomeric separation of arylpropionic acid drugs were systematically developed by reversed phase-high performance liquid chromatography (RP-HPLC) using cellulose-tris-(4-methylbenzoate) (CTMB) as chiral stationary phase (CSP). The effects of the composition of the mobile phase, additives and temperature on chiral separation of flurbiprofen, pranoprofen, naproxen, ibuprofen and loxoprofen were further investigated. The enantiomers had been successfully separated on CSP of CTMB by the mobile phase of methanol-0.1% (v/v) formic acid except naproxen by acetonitrile-0.1% (v/v) formic acid at 25 °C. The mechanisms of the racemic resolution for the above mentioned five drugs are discussed thermodynamically and structurally. The resolutions between respective enantiomers for arylpropionic acid drugs on CTMB had significant differences due to their chromatographic behaviors. The order of resolutions ranked pranoprofen, loxoprofen, flurbiprofen, ibuprofen and naproxen. The method established has been successfully applied to the determination of the enantiomers of the five drugs in commercial preparations under the optimized conditions. It proved that the method is simple, reliable and accurate.

  15. Insights into chiral recognition mechanisms in supercritical fluid chromatography V. Effect of the nature and proportion of alcohol mobile phase modifier with amylose and cellulose tris-(3,5-dimethylphenylcarbamate) stationary phases.

    PubMed

    Khater, Syame; West, Caroline

    2014-12-19

    In enantioselective supercritical fluid chromatography (SFC) with chiral stationary phases (CSP), the elution strength of carbon dioxide is usually modulated by the use of polar organic solvents, also called modifiers. Alcohols like methanol, ethanol and isopropanol are the most commonly used co-solvents. While most applications of chiral SFC are optimized through a process of varying the co-solvent nature and proportion, only a limited number of thorough investigations have been carried out to unravel the effects of the co-solvent on the enantioseparation process. In an attempt to clarify the effect of the mobile phase co-solvent on enantioselective SFC separations, a wide range of compounds (achiral and chiral) were analyzed on an amylosic (Chiralpak AD-H) and a cellulosic (Lux cellulose-1) CSP. The influence of the modifier polarity and steric hindrance must be considered thus several different alcoholic solvents were evaluated: methanol, ethanol, 1-propanol, 2-propanol and 1-butanol, with a proportion of 10% in carbon dioxide. A selected group of racemates was further analyzed with varying proportions of each alcohol ranging from 5 to 25%. Besides, because mixtures of solvents were sometimes reported to produce unexpected results, a 50:50 mixture of methanol and ethanol was also evaluated. Chemometric methods provide some insight into the enantio-separation process and help identifying the differences between the mobile phase conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Evaluation of the chiral recognition properties and the column performances of three chiral stationary phases based on cellulose for the enantioseparation of six dihydropyridines by high-performance liquid chromatography.

    PubMed

    Yu, Jia; Tang, Jing; Yuan, Xiaowei; Guo, Xingjie; Zhao, Longshan

    2017-03-28

    Separations of six dihydropyridine enantiomers on three commercially available cellulose-based chiral stationary phases (Chiralcel OD-RH, Chiralpak IB, and Chiralpak IC) were evaluated with high-performance liquid chromatography (HPLC). The best enantioseparation of the six chiral drugs was obtained with a Chiralpak IC (250 × 4.6 mm i.d., 5 μm) column. Then the influence of the mobile phase including an alcohol-modifying agent and alkaline additive on the enantioseparation were investigated and optimized. The optimal mobile phase conditions and maximum resolution for every analyte were as follows respectively: n-hexane/isopropanol (85:15, v/v) for nimodipine (R = 5.80) and cinildilpine (R = 5.65); n-hexane/isopropanol (92:8, v/v) for nicardipine (R = 1.76) and nisoldipine (R = 1.92); and n-hexane/isopropanol/ethanol (97:2:1, v/v/v) for felodipine (R = 1.84) and lercanidipine (R = 1.47). Relative separation mechanisms are discussed based on the separation results, and indicate that the achiral parts in the analytes' structure showed an important influence on the separation of the chiral column.

  17. Online solid phase extraction liquid chromatography using bonded zwitterionic stationary phases and tandem mass spectrometry for rapid environmental trace analysis of highly polar hydrophilic compounds - Application for the antiviral drug Zanamivir.

    PubMed

    Lindberg, Richard H; Fedorova, Ganna; Blum, Kristin M; Pulit-Prociak, Jolanta; Gillman, Anna; Järhult, Josef; Appelblad, Patrik; Söderström, Hanna

    2015-08-15

    Zanamivir (Za) is a highly polar and hydrophilic antiviral drug used for the treatment of influenza A viruses. Za has been detected in rivers of Japan and it's environmental occurrence has the risk of inducing antiviral resistant avian influenza viruses. In this study, a rapid automated online solid phase extraction liquid chromatography method using bonded zwitterionic stationary phases and tandem mass spectrometry (SPE/LC-MS/MS) for trace analysis of Za was developed. Furthermore, an internal standard (IS) calibration method capable of quantifying Za in Milli-Q, surface water, sewage effluent and sewage influent was evaluated. Optimum pre-extraction sample composition was found to be 95/5 v/v acetonitrile/water sample and 1% formic acid. The developed method showed acceptable linearities (r(2)≥0.994), filtration recovery (≥91%), and intra-day precisions (RSD≤16%), and acceptable and environmentally relevant LOQs (≤20ngL(-1)). Storage tests showed no significant losses of Za during 20 days and +4/-20°C (≤12%) with the exception of influent samples, which should be kept at -20°C to avoid significant Za losses. The applicability of the method was demonstrated in a study on phototransformation of Za in unfiltered and filtered surface water during 28 days of artificial UV irradiation exposure. No significant (≤12%) phototransformation was found in surface water after 28 days suggesting a relatively high photostability of Za and that Za should be of environmental concern.

  18. Immobilized strychnine as a new chiral stationary phase for HPLC.

    PubMed

    Sýkora, David; Vozka, Jiří; Tesařová, Eva; Kalíková, Květa; Havlík, Martin; Matějka, Pavel; Král, Vladimír

    2017-08-01

    A new ion-exchanger type chiral stationary phase for high-performance liquid chromatography was prepared. The synthetic protocol is based on derivatization of silica with (3-iodopropyl)trimethoxysilane in the first step followed by immobilization of strychnine via quaternization of nitrogen atom of the alkaloid strychnine. The synthesized chiral stationary phase was chromatographically characterized. The main effort was headed towards the evaluation of the enantioselectivity of the novel sorbent. For that purpose a set of suitable chiral probes, specifically, binaphthyl derivatives, was employed. The influence of methanol content, concentration of aqueous ammonium acetate buffer, and its pH on retention factors, separation selectivity, and resolution of the atropoisomers of the mentioned chiral solutes was studied in detail. It was demonstrated that the new chiral stationary phase was capable to separate atropoisomers of four out of seven testing compounds. Despite the strong influence of the above mentioned variables on retention, their impact on selectivity and resolution was rather moderate. Concerning retention mechanism, it seems that electrostatic interaction between the positively charged quaternary nitrogen of the chiral stationary phase and anionic solute participates significantly in the retention process. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Separation Differences Among Phenyl Hydride, UDC Cholesterol and Bidentate C8 Stationary Phases for Stability Indicating Methods of Tetracyclines: Journal of Liquid Chromatography & Related Technologies.

    PubMed

    Young, Joshua E; Matyska, Maria T; Azad, Anil K; Yoc, Sergio E; Pesek, Joseph J

    2013-04-01

    Formulation extracts of tetracycline hydrochloride (HCl), minocycline hydrochloride (HCl), and doxycycline hyclate were degraded by strong acidic conditions and heating. Subsequently, components of the extracts were separated by Bidentate C8, Phenyl Hydride and Cholesterol (UDC) HPLC columns operating in the reverse phase mode. The Phenyl Hydride column was able to baseline separate minocycline from the observed degradant, while partial or total co-elution was observed with the other two columns using otherwise identical method conditions. For both the degraded tetracycline HCl and doxycycline hyclate extracts, the UDC column gave the best resolution for the critical pair. The findings suggest that the postulated secondary retention mechanisms of π-π interactions from the Phenyl Hydride and shape selectivity from the UDC can provide superior resolution for structurally similar analytes compared to hydrophobic interactions alone.

  20. Systematic comparison of sensitivity between hydrophilic interaction liquid chromatography and reversed phase liquid chromatography coupled with mass spectrometry.

    PubMed

    Periat, Aurélie; Boccard, Julien; Veuthey, Jean-Luc; Rudaz, Serge; Guillarme, Davy

    2013-10-18

    Hydrophilic interaction liquid chromatography (HILIC) appears as a promising strategy to increase sensitivity with electrospray ionization source (ESI/MS). In the present study, peak heights, background noises and signal-to-noise ratios (S/N) obtained with HILIC-MS/MS and RPLC-MS/MS conditions were systematically compared using a dataset of 56 basic drugs possessing diverse physico-chemical properties. Various mobile phase conditions were investigated, including different pH (3 and 6 in HILIC; 3, 6 and 9 in RPLC) and flow rates (300, 600 and 1000μL/min). The average gain in sensitivity obtained between HILIC and RPLC was equal to 7 and 10 at pH 3 and 6, respectively. However, this value was not reliable, since it was altered by a few compounds possessing an "extreme" behaviour (gain in sensitivity from 100-fold to >8000-fold better). Then, the median gain in sensitivity, equal to 4 in our case, whatever the pH, should be considered. For about 90% of the tested compounds and analytical conditions, the best S/N was systematically attained under HILIC mode. Thanks to PCA representation, it was shown that the basic compounds with pKa between 6 and 8 generally had the best sensitivity in HILIC at pH 6, while the best sensitivity for basic analytes possessing pKa higher than 8 was usually obtained in HILIC at pH 3. As previously reported, the sensitivity gain in HILIC vs. RPLC was explained by the difference in acetonitrile concentration at elution (in average 29% ACN in RPLC and 82% ACN in HILIC at pH 6) leading to better analytes' desolvation. However, it seems that this high proportion of solvent also favourably influenced the ionization by modifying pH and pKa. Indeed the weakest bases of our training set of compounds (pKa between 2 and 5) showed an unexpectedly strong gain in sensitivity, between 20 and 100-fold in comparison to RPLC. These results prove that the ionic character of analytes in solution (i.e., pKa and pH) and the ionization mechanism (i.e., proton

  1. Antibody-ligand interactions for hydrophobic charge-induction chromatography: a surface plasmon resonance study.

    PubMed

    Cheng, Fang; Li, Ming-Yang; Wang, Han-Qi; Lin, Dong-Qiang; Qu, Jing-Ping

    2015-03-24

    This article describes the use of surface plasmon resonance (SPR) spectroscopy to study antibody-ligand interactions for hydrophobic charge-induction chromatography (HCIC) and its versatility in investigating the surface and solution factors affecting the interactions. Two density model surfaces presenting the HCIC ligand (mercapto-ethyl-pyridine, MEP) were prepared on Au using a self-assembly technique. The surface chemistry and structure, ionization, and protein binding of such model surfaces were characterized by X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS), contact-angle titration, and SPR, respectively. The influences of the surface and solution factors, e.g., ligand density, salt concentration, and solution pH, on protein adsorption were determined by SPR. Our results showed that ligand density affects both equilibrium and dynamic aspects of the interactions. Specifically, a dense ligand leads to an increase in binding strength, rapid adsorption, slow desorption, and low specificity. In addition, both hydrophobic interactions and hydrogen bonding contribute significantly to the protein adsorption at neutral pH, while the electrostatic repulsion is overwhelmed under acidic conditions. The hydrophobic interaction at a high concentration of lyotropic salt would cause drastic conformational changes in the adsorbed protein. Combined with the self-assembly technique, SPR proves to be a powerful tool for studying the interactions between an antibody and a chromatographic ligand.

  2. Stationary black diholes

    NASA Astrophysics Data System (ADS)

    Manko, V. S.; Rabadán, R. I.; Sanabria-Gómez, J. D.

    2014-03-01

    In this paper, we present and analyze the simplest physically meaningful model for stationary black diholes—a binary configuration of counterrotating Kerr-Newman black holes endowed with opposite electric charges—elaborated in a physical parametrization on the basis of one of the Ernst-Manko-Ruiz equatorially antisymmetric solutions of the Einstein-Maxwell equations. The model saturates the Gabach-Clement inequality for interacting black holes with struts, and in the absence of rotation, it reduces to the Emparan-Teo electric dihole solution. The physical characteristics of each dihole constituent satisfy identically the well-known Smarr's mass formula.

  3. Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow.

    PubMed

    Staples, Gregory O; Naimy, Hicham; Yin, Hongfeng; Kileen, Kevin; Kraiczek, Karsten; Costello, Catherine E; Zaia, Joseph

    2010-01-15

    Heparan sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. High-performance liquid chromatography (HPLC)-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of postcolumn makeup flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding the oligosaccharide profile, degree of sulfation, and nonreducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.

  4. Reinforcement of frontal affinity chromatography for effective analysis of lectin-oligosaccharide interactions.

    PubMed

    Hirabayashi, J; Arata, Y; Kasai, K

    2000-08-25

    Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 microm diameter), and packed into a miniature column (e.g., 10 x 4.0 mm, 0.126 ml). Twelve pyridylaminated oligosaccharides were applied to the column through a 2-ml sample loop, and their elution patterns were monitored by fluorescence. The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V0); and the dissociation constant, Kd, was calculated according to the literature [K. Kasai, Y. Oda, M. Nishikawa, S. Ishii, J. Chromatogr. 376 (1986) 33]. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The relative affinity of various galectins for asialofetuin could be easily compared in terms of the extent of retardation. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 microg/ml for protein) analyses of lectin-carbohydrate interactions. It should become a powerful tool for elucidation of biomolecular interactions, in particular for functional analysis of a large number of proteins that should be the essential issues of post-genome projects.

  5. Extracting stationary segments from non-stationary synthetic and cardiac signals

    NASA Astrophysics Data System (ADS)

    Rodríguez, María. G.; Ledezma, Carlos A.; Perpiñán, Gilberto; Wong, Sara; Altuve, Miguel

    2015-01-01

    Physiological signals are commonly the result of complex interactions between systems and organs, these interactions lead to signals that exhibit a non-stationary behaviour. For cardiac signals, non-stationary heart rate variability (HRV) may produce misinterpretations. A previous work proposed to divide a non-stationary signal into stationary segments by looking for changes in the signal's properties related to changes in the mean of the signal. In this paper, we extract stationary segments from non-stationary synthetic and cardiac signals. For synthetic signals with different signal-to-noise ratio levels, we detect the beginning and end of the stationary segments and the result is compared to the known values of the occurrence of these events. For cardiac signals, RR interval (cardiac cycle length) time series, obtained from electrocardiographic records during stress tests for two populations (diabetic patients with cardiovascular autonomic neuropathy and control subjects), were divided into stationary segments. Results on synthetic signals reveal that the non-stationary sequence is divided into more stationary segments than needed. Additionally, due to HRV reduction and exercise intolerance reported on diabetic cardiovascular autonomic neuropathy patients, non-stationary RR interval sequences from these subjects can be divided into longer stationary segments compared to the control group.

  6. Interaction between moving tandem wheels and an infinite rail with periodic supports - Green's matrices of the track method in stationary reference frame

    NASA Astrophysics Data System (ADS)

    Mazilu, Traian

    2017-08-01

    This paper approaches the issue of the interaction between moving tandem wheels and an infinite periodically supported rail and points out at the basic characteristics in the steady-state interaction behaviour and in the interaction in the presence of the rail random irregularity. The rail is modelled as an infinite Timoshenko beam resting on supports which are discretely modelling the inertia of the sleepers and ballast and also the viscoelastic features of the rail pads, the ballast and the subgrade. Green‧s matrices of the track method in stationary reference frame were applied so as to conduct the time-domain analysis. This method allows to consider the nonlinearities of the wheel/rail contact and the Doppler effect. The study highlights certain aspects regarding the influence of the wheel base on the wheels/rail contact forces, particularly at the parametric resonance, due to the coincidence between the wheel/rail natural frequency and the passing frequency and also when the rail surface exhibits random irregularity. It has been shown that the wheel/rail dynamic behaviour is less intense when the wheel base equals integer multiple of the sleeper bay.

  7. Simulation of the chromatographic separation process in HPLC employing suspended-state NMR spectroscopy - comparison of interaction behavior for monomeric and hydride-modified C18 stationary phases.

    PubMed

    Yeman, Helen; Nicholson, Tim; Matyska, Maria T; Pesek, Joseph J; Albert, Klaus

    2013-01-01

    The interactions of different analytes with monomeric and hydride-modified stationary phases have been investigated employing suspended-state NMR spectroscopy. The suspended-state high-resolution/magic-angle-spinning (1)H-NMR spectrum of an analyte in the presence of C(18) SP material shows a splitting into two sets of signals for the analyte molecule. One state reflects a closer interaction between analyte and C(18) -modified surface that results in an upfield shift and broader signal half-widths. This phenomenon suggests that the analyte exists in two environments. We report a systematic approach upon the investigation on the interaction in the interface of analyte, mobile phase, and modified silica through synthesis of differently modified silica with a gradual increase in surface coverage. The determination of the signal half-widths and chemical shifts revealed a relationship between the modification technique of the C(18) SPs and the chromatographic and NMR spectroscopic behavior. Increasing ligand density results in higher shielding of the NMR signals for the analyte in the "adsorbed" state. The measurement of spin-lattice relaxation times T(1) of the analyte molecule correlate NMR parameter together with separation behavior in HPLC. Furthermore, suspended-state and solid-state NMR measurements revealed different alkyl chain mobilities for the monomeric and hydride-modified SPs. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

    PubMed Central

    Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

    2011-01-01

    In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media

  9. Automated hydrophobic interaction chromatography column selection for use in protein purification.

    PubMed

    Murphy, Patrick J M; Stone, Orrin J; Anderson, Michelle E

    2011-09-21

    In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein (1). The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH(4;))(2;)SO(4;)). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) (2). As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter (3). Automated column scouting allows for an efficient approach for determining

  10. Extensive database of liquid phase diffusion coefficients of some frequently used test molecules in reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography.

    PubMed

    Song, Huiying; Vanderheyden, Yoachim; Adams, Erwin; Desmet, Gert; Cabooter, Deirdre

    2016-07-15

    Diffusion plays an important role in all aspects of band broadening in chromatography. An accurate knowledge of molecular diffusion coefficients in different mobile phases is therefore crucial in fundamental column performance studies. Correlations available in literature, such as the Wilke-Chang equation, can provide good approximations of molecular diffusion under reversed-phase conditions. However, these correlations have been demonstrated to be less accurate for mobile phases containing a large percentage of acetonitrile, as is the case in hydrophilic interaction liquid chromatography. A database of experimentally measured molecular diffusion coefficients of some 45 polar and apolar compounds that are frequently used as test molecules under hydrophilic interaction liquid chromatography and reversed-phase conditions is therefore presented. Special attention is given to diffusion coefficients of polar compounds obtained in large percentages of acetonitrile (>90%). The effect of the buffer concentration (5-10mM ammonium acetate) on the obtained diffusion coefficients is investigated and is demonstrated to mainly influence the molecular diffusion of charged molecules. Diffusion coefficients are measured using the Taylor-Aris method and hence deduced from the peak broadening of a solute when flowing through a long open tube. The validity of the set-up employed for the measurement of the diffusion coefficients is demonstrated by ruling out the occurrence of longitudinal diffusion, secondary flow interactions and extra-column effects, while it is also shown that radial equilibration in the 15m long capillary is effective.

  11. Hydrophilic interaction liquid chromatography in the separation of a moderately lipophilic drug from its highly polar metabolites--the cardioprotectant dexrazoxane as a model case.

    PubMed

    Kovaříková, Petra; Stariat, Ján; Klimeš, Jiří; Hrušková, Kateřina; Vávrová, Kateřina

    2011-01-21

    This paper presents a systematic study of the retention behavior of a model bisdioxopiperazine drug, dexrazoxane (DEX) and its three polar metabolites (two single open-ring intermediates-B and C and an EDTA-like active compound ADR-925) on different stationary phases intended for hydrophilic interaction liquid chromatography (HILIC). The main aim was to estimate advantages and limitations of HILIC in the simultaneous analysis of a moderately lipophilic parent drug and its highly polar metabolites, including positional isomers, under MS compatible conditions. The study involved two bare silica columns (Ascentic Express HILIC, Atlantis HILIC) and two stationary phases with distinct zwitterionic properties (Obelisc N and ZIC HILIC). The chromatographic conditions (mobile phase strength and pH, column temperature) were systematically modified to assess their impact on retention and separation of the studied compounds. It was found that the bare silica phases were unable to separate the positional isomers (intermediates B and C), whereas both columns with zwitterionic properties (Obelisc N and ZIC HILIC) were able to separate these structurally very similar compounds. However, only ZIC HILIC phase allowed appropriate separation of DEX and all its metabolites to a base line within a single run. A mobile phase composed of a mixture of ammonium formate (0.5 mM) and acetonitrile (25:75, v/v) was suggested as optimal for the simultaneous analysis of DEX and its metabolites on ZIC HILIC. Thereafter, HILIC-LC-MS analysis of DEX and all its metabolites was performed for the first time to obtain basic data about the applicability of the suggested chromatographic conditions. Hence, this study demonstrates that HILIC could be a viable solution for the challenging analysis of moderately polar parent drug along with its highly polar metabolites including the ability to separate structurally very similar compounds, such as positional isomers. Copyright © 2010 Elsevier B.V. All

  12. Hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents for the separation of nucleosides and nucleotide mono-, di- and triphosphates.

    PubMed

    Mateos-Vivas, María; Rodríguez-Gonzalo, Encarnación; García-Gómez, Diego; Carabias-Martínez, Rita

    2015-10-02

    A fast and efficient method for the simultaneous separation of highly polar compounds, in this case nucleosides and nucleotide mono-, di- and triphosphates, using hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) is proposed. This new separation method revealed the possibilities of the formation of hydrophilic ion-pairing compounds. Three stationary phases (HILIC XBridge-Amide, HILIC-CoreShell and ZIC-HILIC) were assayed for the separation of 20 target analytes, and a detailed study of the composition of the mobile phase was made using different salts at different concentrations in a organic-rich mobile phase. We report that in order to prevent the adsorption of nucleotides on the LC-MS setup and to enhance their retention on the HILIC stationary phase, a mobile phase containing hexafluoro-2-propanol and different cations should be used. Four cations were evaluated: ammonium, diethylammonium, triethylammonium and tetrabutylammonium. The results revealed the formation of an ionic-association compound between the phosphorylated analytes and the cationic ion-pairing reagents, whose retention increased with the polarity of the cationic ion-pairing reagent. HILIC XBridge-Amide was found to be the most suitable column for the separation of these analytes, and the optimized mobile phase consisted of an ACN/UHQ water mixture (3min of isocratic elution using 82:18%, v/v and then a fast gradient from 18% to 22% of water) with 100mM hexafluoro-2-propanol and 50mM diethylamine (w(w)pH 9-w(s)pH 10). In a total analysis time of 8min, good results were achieved in terms of resolution. Under these optimum conditions, a further comprehensive study of the retention mechanism was carried out. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Concerning the interaction of non-stationary cross-flow vortices in a three-dimensional boundary layer

    NASA Technical Reports Server (NTRS)

    Bassom, Andrew P.; Hall, Philip

    1990-01-01

    Recently there has been much work devoted to considering some of the many and varied interaction mechanisms which may be operative in three-dimensional boundary layer flows. This paper is concerned with resonant triads of crossflow vortices. The effects of interactions upon resonant triads is examined where each member of the triad has the property of being linearly neutrally stable so that the importance of the interplay between modes can be relatively easily assessed. Modes within the boundary layer flow above a rotating disc are investigated because of the similarity between this disc flow and many important practical flows and, secondly, because the selected flow is an exact solution of the Navier-Stokes equations which makes its theoretical analysis especially attractive. It is demonstrated that the desired triads of linearly neutrally stable modes can exist within the chosen boundary layer flow. Evolution equations are obtained to describe the development of the amplitudes of these modes once the interaction mechanism is accounted for. It is found that the coefficients of the interaction terms within the evolution equations are, in general, given by quite intricate expressions although some elementary numerical work shows that the evaluation of these coefficients is practicable. The basis of the work lends itself to generalization to more complicated boundary layers, and effects of detuning or non-parallelism could be provided for within the asymptotic framework.

  14. Characterization of a chiral stationary phase by HR/MAS NMR spectroscopy and investigation of enantioselective interaction with chiral ligates by transferred NOE.

    PubMed

    Hellriegel, Christine; Skogsberg, Urban; Albert, Klaus; Lämmerhofer, Michael; Maier, Norbert M; Lindner, Wolfgang

    2004-03-31

    The surface chemistry of a chiral stationary phase (CSP) with a (tert-butyl carbamoyl) quinine selector immobilized on thiol-modified silica has been characterized by (1)H HR/MAS NMR and (29)Si CP/MAS NMR spectroscopy. The mostly well-resolved (1)H signals could be assigned to stem from the surface-bound selector and the latter suggested a bi- and trifunctional silane linkage. Suspended-state NMR spectroscopy thus proved a well-characterized surface chemistry as proposed. To study chiral recognition phenomena in the presence of the CSP, (1)H HR/MAS 2D transfer NOESY investigations in methanol-d(4) have been undertaken with various solutes including N-3,5-dinitrobenzoyl derivatives of leucine (DNB-Leu) and N-acetyl phenylalanine (Ac-Phe). Both (R)- and (S)-enantiomers of DNB-Leu and Ac-Phe interacted with the tBuCQN-CSP as indicated by negative cross-peaks in the trNOESY spectra, while the 2D NOESY of the dissolved solutes in absence of the chiral stationary phase showed positive cross-peaks. The intensities of the trNOE cross-peaks were much stronger for the (S)-enantiomers. This stereoselectivity paralleled the experimental chromatographic behavior, where the (S)-enantiomers revealed stronger binding and retention on the tBuCQN-CSP as well. Hence, we were able to correlate the retention behavior to the trNOE NMR spectroscopic data in a qualitative manner.

  15. Insight into the retention processes of phthalate metabolites on different liquid chromatography stationary phases for the development of improved separation methods.

    PubMed

    Gómara, B; Lebrón-Aguilar, R; González, M J; Quintanilla-López, J E

    2015-12-04

    The retention behavior of nine MPAEs has been studied, using commercial LC columns with octadecylsilane (ODS), phenyl, and amide-type SPs. First, it was found that the use of methanol in the mobile phase is not advisable, because induce a transesterification reaction of MPAEs in the electrospray ion source, regardless of the SP used. On the other hand, different responses were observed when representing the logarithm of retention factors (k) vs. the volume fraction of ACN (φ) in the mobile phase, for the three SPs tested. A quite linear trend was obtained for ODS (at φ values below 0.80) and Phenyl columns. On the contrary, the Amide column shows a striking U-shape trend, typical of both hydrophobic and hydrophilic retention mechanisms. Therefore, the separation process was mainly hydrophobic in the ODS and phenyl SPs, but in the amide-type a dual retention mechanism was found, showing zones with predominant hydrophobic or hydrophilic interactions, depending on both the compound and the experimental conditions. A high content of acetonitrile (>75%) and low concentration of formic acid in the mobile phase promote the hydrophilic separation mechanism for MPAEs on the amide SP. So, this dual separation mechanism can be modulated modifying the pH and content of organic modifier in the mobile phase, allowing greater flexibility to develop improved methods. Taking advantage of this, a separation method was optimized in this amide column using a Box-Wilson Central Composite experimental design, which allows separating the studied MPAEs with a time-saving of around 40% comparing to the conventional phenyl SP.

  16. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    PubMed

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  17. Second virial coefficient determination of a therapeutic peptide by self-interaction chromatography.

    PubMed

    Payne, Robert W; Nayar, Rajiv; Tarantino, Ralph; Del Terzo, Sam; Moschera, John; Di, Jie; Heilman, David; Bray, Brian; Manning, Mark Cornell; Henry, Charles S

    2006-01-01

    Self-interaction of macromolecules has been shown to play an important role in a number of physical processes, including crystallization, solubility, viscosity, and aggregation. Peptide self-interaction is not as well studied as for larger proteins, but should play an equally important role. The osmotic second virial coefficient, B, can be used to quantify peptide and protein self-interaction. B values are typically measured using static light scattering (SLS). Peptides, however, do not scatter enough light to allow such measurements. This study describes the first use of self-interaction chromatography (SIC) for the measurement of peptide B values because SIC does not have the molecular size limitations of SLS. In the present work, SIC was used to measure B for enfuvirtide, a 36-amino acid therapeutic peptide, as a function of salt concentration, salt type, and pH. B was found to correlate strongly with solubility and apparent molecular weight. In general, the solubility of enfuvirtide increases with pH from 6 to 10 and decreases as the salt concentration increases from 0 to 0.5M for three different salts. The effect of peptide concentration on B was also investigated and shown to have a significant effect, but only at high concentrations (>80 mg/mL).

  18. Application of Frontal Affinity Chromatography to Study the Biomolecular Interactions with Trypsin.

    PubMed

    Hu, YuanYuan; Qian, Junqing; Guo, Hui; Jiang, ShengLan; Zhang, Zheng

    2015-07-01

    Trypsin is a serine protease that has been proposed as a potential therapeutic target for metabolic disorders and malignancy diseases, thus the identification of biomolecular interactions of compounds to trypsin could be of great therapeutic importance. In this study, trypsin was immobilized on a monolithic silica capillary column via sol-gel. The binding properties of four small molecules (daidzin, genistin, matrine and oxymatrine) to trypsin were examined using the trypsin affinity columns by frontal analysis. The results indicate that the matrine (dissociation constant, Kd = 7.904 μM) has stronger interaction with trypsin than the oxymatrine (Kd = 8.204 μM), whereas daidzin and genistin were nearly have no affinity with trypsin. The results demonstrated that the frontal affinity chromatography can be used for the direct determination of protein-protease inhibitor binding interactions and have several significant advantages, including easy fabricating, reproducible, minimal technological requirements and potential to become a reliable alternative for quantitative studies of biomolecular interactions.

  19. [Preparation and evaluation of N-acryloyltris (hydroxymethyl) aminomethane-bonded chromatographic stationary phase].

    PubMed

    Cheng, Xiaodong; Feng, Yuqi

    2015-09-01

    The present study described the preparation of N-acryloyltris (hydroxymethyl) aminomethane-bonded silica (NAS) stationary phase based on "thiol-ene" click chemistry. The composition of the surface grafts of NAS stationary phase was determined by elemental analysis and the results demonstrated the successful introduction of the N-acryloyltris (hydroxymethyl) aminomethane groups to the silica surface. Similar elemental composition of three batches of the NAS stationary phases exhibited good reproducibility of the preparation strategy. A set of standard compounds were employed to investigate the retention mechanism of the NAS stationary phase by three different empirical equations. The results indicated the retention of the tested analytes on the NAS stationary phase was based more on a mixed-mechanism rather than a simple partitioning or adsorption process. Eight compounds were selected to study the hydrophobic and hydrophilic properties of the NAS stationary phase in mobile phase with different ACN contents. Due to its hydrophilic triolacrylamide groups and short hydrophobic alkyl chains, the NAS phase was successfully applied in both reversed-phase liquid chromatography (RPLC) mode and hydrophilic interaction liquid chromatography (HILIC) mode. The influence of flow rate on the column efficiency was compared in these two modes. In contrast to RPLC columns, the overall heights equivalent to a theoretical plate (HETP) in HILIC is weakly dependent on the retention of the analyte and the HETP curve is much flatter in RPLC than in HILIC at larger reduced velocities. Furthermore, the separation of alkylbenzenes, nucleosides and nucleobases, water-soluble vitamins was achieved on the new stationary phase, demonstrating the excellent application potential.

  20. Analysis of amprolium by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Martínez-Villalba, Anna; Moyano, Encarnación; Galceran, M Teresa

    2010-09-10

    We present a fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of the coccidiostat amprolium in food samples. Tandem mass spectrometry in a triple quadrupole was used for quantitative purposes, and the information from multiple-stage mass spectrometry in an ion-trap mass analyzer contributed to fragmentation studies. Hydrophilic interaction liquid chromatography (HILIC) in a Fused-Core column using isocratic elution (acetonitrile:formic acid/ammonium formate buffer pH 4, 50 mM (60:40)) successfully analyzed this compound in less than 3 min. The HILIC system was coupled to heated electrospray-MS/MS using highly selective-selected reaction monitoring (H-SRM) to improve sensitivity and selectivity for the analysis of amprolium, after a simple sample treatment based on an "extract and shoot" strategy. Accurate mass measurements were performed to identify the interfering compound responsible for causing matrix ion enhancement in the signal of amprolium. The addition of l-carnitine (the interfering compound) (1 microg L(-1)) to standards and sample extracts allowed the use of the external calibration method for quantitative purposes. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation, RSD, lower than 13%), accuracy and linearity and allowed the determination of amprolium down to the ppb level (LODs between 0.1 and 0.6 microg kg(-1)).

  1. Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down proteomics.

    PubMed

    Xiu, Lichen; Valeja, Santosh G; Alpert, Andrew J; Jin, Song; Ge, Ying

    2014-08-05

    One of the challenges in proteomics is the proteome's complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard protein mixtures and a complex cell lysate. Subsequently, the separated intact proteins were identified by high-resolution top-down MS. For the first time, these results have shown the high potential of HIC as a high-resolution protein separation method for top-down proteomics.

  2. Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode.

    PubMed

    To, Brian C S; Lenhoff, Abraham M

    2011-01-21

    The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.

  3. Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography.

    PubMed

    Guo, Xiujie; Zhang, Xiuli; Feng, Jiatao; Guo, Zhimou; Xiao, Yuansheng; Liang, Xinmiao

    2013-04-01

    Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C18 columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C18 column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C18 and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C18 column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products.

  4. Determination of major phlorotannins in Eisenia bicyclis using hydrophilic interaction chromatography: seasonal variation and extraction characteristics.

    PubMed

    Kim, Sang Min; Kang, Suk Woo; Jeon, Je-Seung; Jung, Yu-Jin; Kim, Woo-Ri; Kim, Chul Young; Um, Byung-Hun

    2013-06-15

    In this study, a hydrophilic interaction chromatography (HILIC) condition was developed for the simultaneous determination of five major phlorotannins from an extract of Eisenia bicyclis (Kjellman) Setchell with good linearity (r(2)>0.999). Based on this method, the seasonal variations and extraction characteristics, in terms of total extraction yield and the content of the phlorotannins, were investigated under various extraction conditions. In results, the yields and phlorotannins were increased two-to-four times in summer (June-October) and then, were decreased to normal levels in winter (November-March). In the extraction of E. bicyclis, ethanol percentage in water, extraction time and washing time significantly affected the yield of the extract and the phlorotannins, whereas the temperature and the sample/solvent ratio impacted the extraction to a lesser degree. These results will be useful information in the application of this macroalga in the commercial areas related to nutraceuticals, pharmaceuticals, and cosmeceuticals.

  5. A simple graphical representation of selectivity in hydrophilic interaction liquid chromatography.

    PubMed

    Ibrahim, Mohammed E A; Liu, Yang; Lucy, Charles A

    2012-10-19

    This paper uses the HILIC selectivity data of Dinh et al. (J. Chromatogr. A 1218 (2011) 5880) to yield simple and easy to understand plots analogous to Neue plots for selectivity in HILIC. The plots categorize 21 previously studied HILIC phases (data from Dinh et al.), 8 additional HILIC columns and 4 reversed phase columns (our data) using selected probes for specific interactions. The relative retention of cytosine vs. uracil is used to probe the "hydrophilicity" of the HILIC phases; adenosine vs. adenine is used to probe the ability of the stationary phase to participate in hydrogen bonding; and benzyltrimethylammonium (BTMA) vs. cytosine is used to probe the cation exchange and anion exchange character of the column. Plots of kBTMA/kcytosine vs. kcytosine/kuracil successfully classify silica, amide, zwitterionic, diol and reverse phase columns in terms of their HILIC behavior. Polymeric columns including polymer substrate and polymer coated columns show low ion exchange character, but vary widely in their hydrophilicity. Alternatively a HILIC-Phase Selectivity Chart, in analogy to the Neue plot, is constructed by plotting log(kBTMA/kcytosine) vs. log(kcytosine). This plot enables classification of HILIC columns that will yield similar or significantly different separations. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Interaction chromatography for characterization and large-scale fractionation of chemically heterogeneous copolymers

    NASA Astrophysics Data System (ADS)

    Han, Junwon

    The remarkable development of polymer synthesis techniques to make complex polymers with controlled chain architectures has inevitably demanded the advancement of polymer characterization tools to analyze the molecular dispersity in polymeric materials beyond size exclusion chromatography (SEC). In particular, man-made synthetic copolymers that consist of more than one monomer type are disperse mixtures of polymer chains that have distributions in terms of both chemical heterogeneity and chain length (molar mass). While the molecular weight distribution has been quite reliably estimated by the SEC, it is still challenging to properly characterize the chemical composition distribution in the copolymers. Here, I have developed and applied adsorption-based interaction chromatography (IC) techniques as a promising tool to characterize and fractionate polystyrene-based block, random and branched copolymers in terms of their chemical heterogeneity. The first part of this thesis is focused on the adsorption-desorption based purification of PS-b-PMMA diblock copolymers using nanoporous silica. The liquid chromatography analysis and large scale purification are discussed for the PS-b-PMMA block copolymers that have been synthesized by sequential anionic polymerization. SEC and IC are compared to critically analyze the contents of PS homopolymers in the as-synthesized block copolymers. In addition, I have developed an IC technique to provide faster and more reliable information on the chemical heterogeneity in the as-synthesized block copolymers. Finally, a large scale (multi-gram) separation technique is developed to obtain "homopolymer-free" block copolymers via a simple chromatographic filtration technique. By taking advantage of the large specific surface area of nanoporous silica (≈300m 2/g), large scale purification of neat PS-b-PMMA has successfully been achieved by controlling adsorption and desorption of the block copolymers on the silica gel surface using a

  7. ANALYSIS OF DRUG INTERACTIONS WITH HIGH DENSITY LIPOPROTEIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Sike; Sobansky, Matthew R.; Hage, David S.

    2009-01-01

    Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography. This approach was evaluated by using it to examine the binding of high density lipoprotein (HDL) to the drugs propranolol or verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPLC columns with reproducible binding to propranolol over four to five days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interactions were occurring between R/S-propranolol and HDL at 37°C: saturable binding with an association equilibrium constant (Ka) of 1.1–1.9 × 105 M−1, and non-saturable binding with an overall affinity constant (n Ka) of 3.7–4.1 × 104 M−1. Similar results were found at 4 and 27°C. Verapamil also gave similar behavior, with a Ka of 6.0 × 104 M−1 at 37°C for the saturable sites and a n Ka value for the non-saturable sites of 2.5 × 104 M−1. These measured affinities gave good agreement with solution-phase values. The results indicated HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body. PMID:19833090

  8. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  9. Accurate prediction of retention in hydrophilic interaction chromatography (HILIC) by back calculation of high pressure liquid chromatography (HPLC) gradient profiles.

    PubMed

    Wang, Nu; Boswell, Paul G

    2017-08-26

    Gradient retention times are difficult to project from the underlying retention factor (k) vs. solvent composition (φ) relationships. A major reason for this difficulty is that gradients produced by HPLC pumps are imperfect - gradient delay, gradient dispersion, and solvent mis-proportioning are all difficult to account for in calculations. However, we recently showed that a gradient "back-calculation" methodology can measure these imperfections and take them into account. In RPLC, when the back-calculation methodology was used, error in projected gradient retention times is as low as could be expected based on repeatability in the k vs. φ relationships. HILIC, however, presents a new challenge: the selectivity of HILIC columns drift strongly over time. Retention is repeatable in short time, but selectivity frequently drifts over the course of weeks. In this study, we set out to understand if the issue of selectivity drift can be avoid by doing our experiments quickly, and if there any other factors that make it difficult to predict gradient retention times from isocratic k vs. φ relationships when gradient imperfections are taken into account with the back-calculation methodology. While in past reports, the accuracy of retention projections was >5%, the back-calculation methodology brought our error down to ∼1%. This result was 6-43 times more accurate than projections made using ideal gradients and 3-5 times more accurate than the same retention projections made using offset gradients (i.e., gradients that only took gradient delay into account). Still, the error remained higher in our HILIC projections than in RPLC. Based on the shape of the back-calculated gradients, we suspect the higher error is a result of prominent gradient distortion caused by strong, preferential water uptake from the mobile phase into the stationary phase during the gradient - a factor our model did not properly take into account. It appears that, at least with the stationary phase

  10. Simultaneous analysis of acetaminophen, p-aminophenol and aspirin metabolites by hydrophilic interaction and strong anion exchange capillary liquid chromatography coupled to amperometric detection.

    PubMed

    Zheng, Minmin; Wu, Yimin; Lu, Lanxiang; Ding, Kang; Tang, Fengxiang; Lin, Zian; Wu, Xiaoping

    2011-08-01

    A simple and sensitive method has been developed for the simultaneous determination of polar nonsteroidal pharmaceuticals and metabolites, including acetaminophen, p-aminophenol and several aspirin metabolites (salicylic acid, gentisic acid, salicyluric acid and 2,3-dihydroxybenzoic acid), by capillary liquid chromatography with amperometric detection. Using a capillary monolithic column with mixed mode stationary phases and a mobile phase composed of acetonitrile and Tris buffer, rapid separation of six polar analytes was achieved within 8 min, and a hydrophilic interaction and strong anion exchange separation mechanism were exhibited. Method detection limits of six analytes ranged from 10 to 50 ng/mL. In terms of precision, the intra- and interday relative standard deviation values in all analytes never exceeded 3.1% for migration time and 8.9% for peak areas, respectively. This method provided a simple, rapid and cost-effective approach for the analysis of polar pharmaceuticals. The applicability of the method in pharmacokinetics was verified by spiking human serum samples with the compounds and analyzing the recoveries. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry.

    PubMed

    Mazzarino, Monica; Fiacco, Ilaria; de la Torre, Xavier; Botrè, Francesco

    2011-11-11

    The chromatographic behaviour of 44 polar compounds (23 beta-adrenergic agents, 11 stimulants, 4 narcotics and 6 phenolalkylamines) included in the list of prohibited substances and methods of the World Anti-Doping Agency, has been investigated under hydrophilic interaction liquid chromatography conditions by application of different mobile phase compositions (percentage of the organic solvent, type and amount of mobile phase additive and ionic strength) and column temperatures. Detection of analytes was performed by a triple quadrupole mass spectrometer in positive ionization mode and selected reaction monitoring acquisition mode after liquid/liquid extraction. Data collected using as stationary phase type-B silica materials from different producers, showed that the best chromatographic conditions in terms of peak shape, selectivity and chromatographic retention were obtained using an initial percentage of acetonitrile of 90%, a column temperature of 35 °C, a mobile phase pH of 4.5 and ammonium acetate (5 mM) and acetic acid (0.1%) as mobile phase additives. The selected chromatographic conditions were used to develop screening and confirmation analytical procedures to detect polar compounds in human urine for antidoping purpose. The developed methods were validated in terms of specificity, matrix effect, linearity, precision, accuracy, sensitivity, robustness and repeatability of retention times and relative ion abundances. Such methods offer attractive alternatives and considerable advantages over traditional approaches especially for the analysis of the phenolalkylamines. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Separation and analysis of mono-glucosylated lipids in brain and skin by hydrophilic interaction chromatography based on carbohydrate and lipid moiety.

    PubMed

    Nakajima, Kazuki; Akiyama, Hisako; Tanaka, Kaori; Kohyama-Koganeya, Ayako; Greimel, Peter; Hirabayashi, Yoshio

    2016-09-15

    Mono-glycosylated sphingolipids and glycerophospholipids play important roles in diverse biological processes and are linked to a variety of pathologies, such as Parkinson disease. The precise identification of the carbohydrate head group of these lipids is complicated by their isobaric nature and by substantial differences in concentration in different biological samples. To overcome these obstacles, we developed a zwitterionic (ZIC)-hydrophilic interaction chromatography (HILIC) electrospray ionization tandem mass spectrometry method. ZIC-HILIC preferentially retains inositol, followed by glucose- and galactose-featuring lipids. Comparison with unmodified silica gel HILIC stationary phase revealed different retention specificity. To evaluate the precision of ZIC-HILIC, we quantified glucosyl- (GlcCer) and galactosylceramides (GalCer) in seven different regions of the mouse brain and discovered that GlcCer and GalCer concentrations are inversely related. The highest GalCer (lowest GlcCer) content was found in the medulla oblongata and hippocampus, whereas the highest GlcCer (lowest GalCer) content was found in other regions. With a neutral loss scan, ZIC-HILIC resolved glucosylceramide species featuring non-hydroxylated fatty acid, hydroxylated fatty acid, and trihydroxy sphingoid bases in mouse epidermis samples. This demonstrates that our ZIC-HILIC-based approach is a valuable tool for characterizing the structural diversity of mono-glucosylated lipids in biological material and for quantifying these important lipids. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Interactions between orographic gravity wave drag and forced stationary planetary waves in the winter northern hemisphere middle atmosphere

    SciTech Connect

    McLandress, C. ); McFarlane, N.A. )

    1993-07-01

    A quasigeostrophic model is used to study the combined interaction among orographically generated gravity wave drag, forced planetary waves, and zonal mean flows in the Northern Hemisphere winter stratosphere and mesosphere. The localized gravity wave drag is shown to generate planetary waves in the mesosphere that, in turn, exert a substantial drag on the zonal mean flow via the Eliassen-Palm flux divergence. The amount of planetary wave drag is found to depend not only on the presence of the localized source of orographic gravity wave drag but also on the presence of upward-propagating planetary waves in the lower stratosphere. The zonal mean wind field exhibits a split jet structure with the larger jet maximum situated in the upper stratosphere at 30[degrees]N. This feature is shown to arise from the presence of weak winds above the subtropical tropospheric jet maximum, which results in a region of low-level gravity wave breaking and reduced drag and larger winds above. 33 refs., 18 figs.

  14. Time-resolved Tomo-PIV measurements of the interaction between a stationary held sphere and a turbulent boundary layer.

    NASA Astrophysics Data System (ADS)

    van Hout, Rene; Eisma, Jerke; Overmars, Edwin; Elsinga, Gerrit; Westerweel, Jerry

    2015-11-01

    Time resolved tomographic PIV measurements (acquisition rate 250Hz) were performed in a turbulent boundary layer (TBL) on the side wall of an open channel, water flow facility (cross section 60x60cm, Wx H) , 3.5m downstream of the inlet at a bulk flow velocity of Ub = 0.17m/s (Reb =Ub H / ν = 102x103, δ0 . 99 = 5 . 0 cm, Reθ = 891). The measurement volume was a horizontal slab (6x1.5x6cm3, lx wx h) extending from the side wall, 30cm above the bottom. The Tomo-PIV setup comprised four high-speed ImagerPro HS cameras (2016x2016pixels), a high-speed laser (Nd:YLF, Darwin Duo 80M, Quantronix), optics/prisms and data acquisition/processing software (LaVision, DaVis8.2). A sphere with diameter, D = 6mm (D+ = 51, ``+'' denotes inner wall scaling), was positioned at y = 37.5 and 5.4mm (y+ = 319 and 46) from the wall (measured from the sphere's center). The latter position covers most of the buffer layer while the former is well in the outer layer. Sphere Reynolds numbers based on D and the average streamwise velocity at the sphere's center were 984 (y+ = 319) and 684 (y+ = 46). Results show the interaction between the coherent turbulence structures in the TBL and those generated in the sphere's wake. Total and partial destruction of the log-law layer is observed when the sphere is positioned in the buffer and outer layer, respectively.

  15. Comprehensive analysis of pharmaceutical products using simultaneous mixed-mode (ion-exchange/reversed-phase) and hydrophilic interaction liquid chromatography.

    PubMed

    Kazarian, Artaches A; Nesterenko, Pavel N; Soisungnoen, Phimpha; Burakham, Rodjana; Srijaranai, Supalax; Paull, Brett

    2014-08-01

    Liquid chromatographic assays were developed using a mixed-mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve-mediated column switching and was based upon a single high-performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion-exchange, (ii) mixed-mode interactions under an applied dual gradient (reversed-phase/ion-exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C18 column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed-mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well-resolved unknown peaks. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies

    PubMed Central

    Estep, Patricia; Caffry, Isabelle; Yu, Yao; Sun, Tingwan; Cao, Yuan; Lynaugh, Heather; Jain, Tushar; Vásquez, Maximiliano; Tessier, Peter M; Xu, Yingda

    2015-01-01

    The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties. PMID:25790175

  17. Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides.

    PubMed

    Philibert, Gwenaëlle S; Olesik, Susan V

    2011-11-11

    Hydrophilic interaction chromatography (HILIC) is a liquid chromatographic separation mechanism commonly used for polar biological molecules. The use of enhanced-fluidity liquid chromatography (EFLC) with mixtures of methanol/water/carbon dioxide is compared to acetonitrile/water mobile phases for the separation of nucleosides and nucleotides under HILIC conditions. Enhanced-fluidity liquid chromatography involves using common mobile phases with the addition of substantial proportions of a dissolved gas which provides greater mobile phase diffusivity and lower viscosity. The impact of varying several experimental parameters, including temperature, addition of base, salt, and CO₂ was studied to provide optimized HILIC separations. Each of these parameters plays a key role in the retention of the analytes, which demonstrates the complexity of the retention mechanism in HILIC. The tailing of phosphorylated compounds was overcome with the use of phosphate salts and the addition of a strong base; efficiency and peak asymmetry were compared with the addition of either triethylamine (TEA), 1,4-diazabicyclo [2.2.2] octane (DABCO) or 1,5-diazabicyclo [4.3.0] non-5-ene (DBN). DBN and DABCO both led to increased efficiency and lower peak asymmetry; DBN provided the best results. Sodium chloride and carbon dioxide were added to enhance the selectivity between the analytes, giving a successful isocratic separation of nucleosides and nucleotides within 8 min. The retention mechanism involved in EFL-HILIC was explored by varying the temperature and the mole fraction of CO₂. These studies showed that partitioning was the dominant mechanism. The thermodynamics study confirmed that the solvent strength is maintained in EFLC and that a change in entropy was mainly responsible for the improved selectivity. The selectivity using methanol/water/carbon dioxide varied greatly compared to that obtained with acetonitrile/water. Finally while this study highlights the optimization of EFL

  18. Solute-solvent interactions in micellar electrokinetic chromatography. III. Characterization of the selectivity of micellar electrokinetic chromatography systems.

    PubMed

    Fuguet, Elisabet; Ràfols, Clara; Bosch, Elisabeth; Abraham, Michael H; Rosés, Martí

    2002-01-04

    Several micellar electrokinetic chromatography (MEKC) systems (sodium dodecyl sulfate, lithium dodecyl sulfate, lithium perfluorooctanesulfonate, sodium cholate, sodium deoxycholate, tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide) have been characterized by means of the solvation parameter model. It has been observed that the coefficients of the correlation equations depend strongly on the particular set of compounds analyzed. Principal component analysis has been used to characterize the 2975 compounds with available solute descriptors and to select an appropriate subset of compounds to be analyzed by MEKC. With this set of compounds, the MEKC systems have been characterized. Principal component analysis has also been used to show the similarities and differences between the properties of the surfactants characterized by MEKC.

  19. Characterization of goat colostrum oligosaccharides by nano-liquid chromatography on chip quadrupole time-of-flight mass spectrometry and hydrophilic interaction liquid chromatography-quadrupole mass spectrometry.

    PubMed

    Martín-Ortiz, A; Salcedo, J; Barile, D; Bunyatratchata, A; Moreno, F J; Martin-García, I; Clemente, A; Sanz, M L; Ruiz-Matute, A I

    2016-01-08

    A detailed qualitative and quantitative characterization of goat colostrum oligosaccharides (GCO) has been carried out for the first time. Defatted and deproteinized colostrum samples, previously treated by size exclusion chromatography (SEC) to remove lactose, were analyzed by nanoflow liquid chromatography-quadrupole-time of flight mass spectrometry (Nano-LC-Chip-Q-TOF MS). Up to 78 oligosaccharides containing hexose, hexosamine, fucose, N-acetylneuraminic acid or N-glycolylneuraminic acid monomeric units were identified in the samples, some of them detected for the first time in goat colostra. As a second step, a hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS) methodology was developed for the separation and quantitation of the main GCO, both acidic and neutral carbohydrates. Among other experimental chromatographic conditions, mobile phase additives and column temperature were evaluated in terms of retention time, resolution, peak width and symmetry of target carbohydrates. Narrow peaks (wh: 0.2-0.6min) and good symmetry (As: 0.8-1.4) were obtained for GCO using an acetonitrile:water gradient with 0.1% ammonium hydroxide at 40°C. These conditions were selected to quantify the main oligosaccharides in goat colostrum samples. Values ranging from 140 to 315mgL(-1) for neutral oligosaccharides and from 83 to 251mgL(-1) for acidic oligosaccharides were found. The combination of both techniques resulted to be useful to achieve a comprehensive characterization of GCO.

  20. Characterization of goat colostrum oligosaccharides by nano-liquid chromatography on chip quadrupole time-of-flight mass spectrometry and hydrophilic interaction liquid chromatography-quadrupole mass spectrometry

    PubMed Central

    Martín-Ortiz, A.; Salcedo, J.; Barile, D.; Bunyatratchata, A.; Moreno, F.J.; Martin-García, I.; Clemente, A.; Sanz, M.L.; Ruiz-Matute, A.I.

    2016-01-01

    A detailed qualitative and quantitative characterization of goat colostrum oligosaccharides (GCO) has been carried out for the first time. Defatted and deproteinized colostrum samples, previously treated by size exclusion chromatography (SEC) to remove lactose, were analyzed by nanoflow liquid chromatography-quadrupole-time of flight mass spectrometry (Nano-LC-Chip-Q-TOF MS). Up to 78 oligosaccharides containing hexose, hexosamine, fucose, N-acetylneuraminic acid or N-glycolylneuraminic acid monomeric units were identified in the samples, some of them detected for the first time in goat colostra. As a second step, a hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS) methodology was developed for the separation and quantitation of the main GCO, both acidic and neutral carbohydrates. Among other experimental chromatographic conditions, mobile phase additives and column temperature were evaluated in terms of retention time, resolution, peak width and symmetry of target carbohydrates. Narrow peaks (wh: 0.2–0.6 min) and good symmetry (As: 0.8–1.4) were obtained for GCO using an acetonitrile:water gradient with 0.1% ammonium hydroxide at 40 °C. These conditions were selected to quantify the main oligosaccharides in goat colostrum samples. Values ranging from 140 to 315 mg L−1 for neutral oligosaccharides and from 83 to 251 mg L−1 for acidic oligosaccharides were found. The combination of both techniques resulted to be useful to achieve a comprehensive characterization of GCO. PMID:26427327

  1. Nontargeted lipidomic characterization of porcine organs using hydrophilic interaction liquid chromatography and off-line two-dimensional liquid chromatography-electrospray ionization mass spectrometry.

    PubMed

    Cífková, Eva; Holčapek, Michal; Lísa, Miroslav

    2013-09-01

    Lipids form a significant part of animal organs and they are responsible for important biological functions, such as semi-permeability and fluidity of membranes, signaling activity, anti-inflammatory processes, etc. We have performed a comprehensive nontargeted lipidomic characterization of porcine brain, heart, kidney, liver, lung, spinal cord, spleen, and stomach using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI/MS) to describe the representation of individual lipid classes in these organs. Detailed information on identified lipid species inside classes are obtained based on relative abundances of deprotonated molecules [M-H](-) in the negative-ion ESI mass spectra, which provides important knowledge on phosphatidylethanolamines and their different forms of fatty acyl linkage (ethers and plasmalogens), phosphatidylinositols, and hexosylceramides containing nonhydroxy- and hydroxy-fatty acyls. The detailed analysis of identified lipid classes using reversed-phase liquid chromatography in the second dimension was performed for porcine brain to determine more than 160 individual lipid species containing attached fatty acyls of different acyl chain length, double-bond number, and positions on the glycerol skeleton. The fatty acid composition of porcine organs is determined by gas chromatography with flame ionization detection after the transesterification with sodium methoxide.

  2. Natural terpene derivatives as new structural task-specific ionic liquids to enhance the enantiorecognition of acidic enantiomers on teicoplanin-based stationary phase by high-performance liquid chromatography.

    PubMed

    Flieger, Jolanta; Feder-Kubis, Joanna; Tatarczak-Michalewska, Małgorzata; Płazińska, Anita; Madejska, Anna; Swatko-Ossor, Marta

    2017-06-01

    We present the specific cooperative effect of a semisynthetic glycopeptide antibiotic teicoplanin and chiral ionic liquids containing the (1R,2S,5R)-(-)-menthol moiety on the chiral recognition of enantiomers of mandelic acid, vanilmandelic acid, and phenyllactic acid. Experiments were performed chromatographically on an Astec Chirobiotic T chiral stationary phase applying the mobile phase with the addition of the chiral ionic liquids. The stereoselective binding of enantiomers to teicoplanin in presence of new chiral ionic liquids were evaluated applying thermodynamic measurements and the docking simulations. Both the experimental and theoretical methods revealed that the chiral recognition of enantiomers in the presence of new chiral ionic liquids was enthalpy driven. The changes of the teicoplanin conformation occurring upon binding of the chiral ionic liquids are responsible for the differences in the standard changes in Gibbs energy (ΔG(0) ) values obtained for complexes formed by the R and S enantiomers and teicoplanin. Docking simulations revealed the steric adjustment between the chiral ionic liquids cyclohexane ring (chair conformation) and the β-d-glucosamine ring of teicoplanin and additionally hydrophobic interactions between the decanoic aliphatic chain of teicoplanin and the alkyl group of the tested salts. The obtained terpene derivatives can be considered as "structural task-specific ionic liquids" responsible for enhancing the chiral resolution in synergistic systems with two chiral selectors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Evaluation of beta-lactoglobulin as a stationary phase in high-performance liquid chromatography and as a buffer additive in capillary electrophoresis: observation of a surprising lack of stereoselectivity.

    PubMed

    Massolini, G; De Lorenzi, E; Lloyd, D K; McGann, A M; Caccialanza, G

    1998-08-07

    Previous studies have reported that alpha1-acid glycoprotein is quite similar in amino acid sequence and disulfide bond arrangements to members of a group of proteins which include beta-lactoglobulin (BLG). Since generally homologous proteins retain some similarity in function at the molecular level, we decided to evaluate the enantioselective properties of BLG as an high-performance liquid chromatographic chiral stationary phase (HPLC-CSP), and as an additive in capillary electrophoresis (CE). Two columns with differences in internal diameter and method of immobilisation on epoxide silica were prepared. Chiral acidic, basic and uncharged drugs were chromatographed and mobile phase parameters, namely pH and type of organic modifier, were varied in order to test the column performance. The CE approach has some advantages in that there is no need for immobilisation and only a small amount of protein is required. BLG was therefore tested as a CE buffer additive, using the same analytes as in the HPLC study. Although one would expect that a protein would display some enantioselectivity, BLG did not show any enantioselectivity whatsoever in either system; the protein has fairly weak interaction with the majority of the test solutes, as indicated by both techniques.

  4. Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-glycomics analysis.

    PubMed

    Zhao, Yun; Law, Henry C H; Zhang, Zaijun; Lam, Herman C; Quan, Quan; Li, Guohui; Chu, Ivan K

    2015-10-09

    In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-glycomics analyses. The performance of the 3D system alone was benchmarked through the analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-glycomics analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.

  5. Analytical approach to determining human biogenic amines and their metabolites using eVol microextraction in packed syringe coupled to liquid chromatography mass spectrometry method with hydrophilic interaction chromatography column.

    PubMed

    Konieczna, Lucyna; Roszkowska, Anna; Synakiewicz, Anna; Stachowicz-Stencel, Teresa; Adamkiewicz-Drożyńska, Elżbieta; Bączek, Tomasz

    2016-04-01

    Analysis of biogenic amines (BAs) in different human samples provides insight into the mechanisms of various biological processes, including pathological conditions, and thus may be very important in diagnosing and monitoring several neurological disorders and cancerous tumors. In this work, we developed a simple and fast procedure using a digitally controlled microextraction in packed syringe (MEPS) coupled to liquid chromatography mass spectrometry (LC-MS) method for simultaneous determination of biogenic amines, their precursors and metabolites in human plasma and urine samples. The separation of 12 low molecular weight and hydrophilic molecules with a wide range of polarities was achieved with hydrophilic interaction chromatography (HILIC) column without derivatization step in 12 min. MEPS was implemented using the APS sorbent in semi-automated analytical syringe (eVol(®)) and small volume of urine and plasma samples, 5 0µL and 100 μL, respectively. We evaluated important parameters influencing MEPS efficiency, including stationary phase selection, sample pH and volume, number of extraction cycles, and washing and elution volumes. In optimized MEPS conditions, the analytes were eluted by 3 × 50 μL of methanol with 0.1% formic acid. The chromatographic separation of analytes was performed on XBridge Amide™ BEH analytical column (3.0mm × 100 mm, 3.5 µm) using gradient elution with mobile phase consisting of phase A: 10mM ammonium formate buffer in water pH 3.0 and phase B: 10mM ammonium formate buffer in acetonitrile pH 3.0. The LC-HILIC-MS method was validated and, in optimum conditions, presented good linearity in concentration range within 10-2000 ng/mL for all the analytes with a determination coefficient (r(2)) higher than 0.999 for plasma and urine samples. Method recovery ranged within 87.6-104.3% for plasma samples and 84.2-98.6% for urine samples. The developed method utilizing polar APS sorbent along with polar HILIC column was applied for

  6. Study of Low Molecular Weight Impurities in Pluronic Triblock Copolymers using MALDI, Interaction Chromatography, and NMR

    NASA Astrophysics Data System (ADS)

    Helming, Z.; Zagorevski, D.; Ryu, C. Y.

    2014-03-01

    Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers are a group of commercial macromolecular amphiphilic surfactants that have been widely studied for their applications in polymer-based nanotechnology and drug-delivery. It has been well-established that the synthesis of commercial Pluronic triblocks results in low molecular weight ``impurities,'' which are generally disregarded in the applications and study of these polymers. These species have been shown to have significant effects on the rheological properties of the material, as well as altering the supramolecular ``micellar'' structures for which the polymers are most often used. We have isolated the impurities from the bulk Pluronic triblock using Interaction Chromatography (IC) techniques, and subjected them to analysis by H1 NMR and MALDI (Matrix-Assisted Laser Desorption Ionization) Mass Spectrometry to identify relative block composition and molecular weight information. We report significant evidence of at least two polymeric components: a low-molecular-weight homopolymer of poly(ethylene oxide) and a ``blocky'' copolymer of both poly(ethylene oxide) and poly(propylene oxide). This has significant implications, not only for the applied usage of Pluronic triblock copolymers, but for the general scientific acceptance of the impurities and their effects on Pluronic micelle and hydrogel formation.

  7. Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody.

    PubMed

    Yu, Deqiang; Chen, Xiaonong; Pelton, Robert; Ghosh, Raja

    2008-04-15

    This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.

  8. Determination of ethyl glucuronide in human hair by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Yaldiz, Fadile; Daglioglu, Nebile; Hilal, Ahmet; Keten, Alper; Gülmen, Mete Korkut

    2013-10-01

    Ethyl glucuronide (EtG) is a direct metabolite of ethanol and has been utilized as a marker for alcohol intake. This study presents development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the analysis of EtG in human hair samples. The linearity was assessed in the range of 5-2000 pg/mg hair, with a correlation coefficient of >0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 0.05 pg/mg and 0.18 pg/mg in hair, respectively. Differently from the extraction procedures in the literature, a fast and simple liquid-liquid method was used and highest recoveries and cleanest extracts were obtained. The method was successfully applied to 30 human hair samples which were taken from those who state they consume alcohol. EtG concentrations in the hair samples of alcohol users participated in this study, ranged between 1.34 and 82.73 pg/mg. From the concentration of EtG in hair strands 20 of the 30 subjects can be considered regular moderate drinkers. Copyright © 2013 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  9. Separation of purine and pyrimidine bases and nucleosides by hydrophilic interaction chromatography.

    PubMed

    Marrubini, Giorgio; Mendoza, Bolivar Enrique Castillo; Massolini, Gabriella

    2010-03-01

    The separation of 12 model compounds chosen among purine and pyrimidine bases and nucleosides was studied by using hydrophilic interaction chromatography (HILIC). The compounds investigated were small molecules with relevant properties for biomedical and pharmaceutical studies. The mixture of pyrimidines and purines was applied on a ZIC-HILIC 150 x 2.1 mm, 5 microm, and two TSKgel Amide-80 150 x 2.0 mm, 5 microm and 3 microm particle size columns. The retention of the analytes was studied by varying ACN%, ammonium formate concentration, pH, and column temperature. The results obtained confirmed the elution order of nucleobases, nucleosides, and nucleotides based on their hydrophobicity. The retention mechanism of the columns was studied considering the models used for describing partitioning and surface adsorption. The influence on retention of chromatographic conditions (ACN%, salt concentration, pH, and temperature) was described and discussed for both columns. The optimization of the conditions studied allowed to assess a gradient method for the separation of the 12 analytes. The developed method is a valuable alternative to existing methods for the separation of the compounds concerned.

  10. ANALYSIS OF DRUG INTERACTIONS WITH VERY LOW DENSITY LIPOPROTEIN BY HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Sobansky, Matthew R.; Hage, David S.

    2014-01-01

    High-performance affinity chromatography (HPAC) was utilized to examine the binding of very low density lipoprotein (VLDL) with drugs, using R/S-propranolol as a model. These studies indicated that two mechanisms existed for the binding of R- and S-propranolol with VLDL. The first mechanism involved non-saturable partitioning of these drugs with VLDL, which probably occurred with the lipoprotein's non-polar core. This partitioning was described by overall affinity constants of 1.2 (± 0.3) × 106 M-1 for R-propranolol and 2.4 (± 0.6) × 106 M-1 for S-propranolol at pH 7.4 and 37 °C. The second mechanism occurred through saturable binding by these drugs at fixed sites on VLDL, such as represented by apolipoproteins on the surface of the lipoprotein. The association equilibrium constants for this saturable binding at 37 °C were 7.0 (± 2.3) × 104 M-1 for R-propranolol and 9.6 (± 2.2) × 104 M-1 for S-propranolol. Comparable results were obtained at 20 °C and 27 °C for the propranolol enantiomers. This work provided fundamental information on the processes involved in the binding of R- and S-propranolol to VLDL, while also illustrating how HPAC can be used to evaluate relatively complex interactions between agents such as VLDL and drugs or other solutes. PMID:25103529

  11. Investigation of the interaction of Mercurochrome constituents with proteins using liquid chromatography/mass spectrometry.

    PubMed

    Wilken, Andrea; Janzen, Rasmus; Holtkamp, Michael; Nowak, Sascha; Sperling, Michael; Vogel, Martin; Karst, Uwe

    2010-08-01

    The interaction of Mercurochrome, a medical preparation based on the mercury organic compound merbromin, with free thiols in low molecular weight peptides and in proteins has been investigated by means of liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS). Beta-lactoglobulin A (beta-LGA) from bovine milk (18.4 kDa) has been used as the model protein. It was found that, in contrast to assumptions in literature, the commercial product itself is a heterogeneous mixture of moderate chemical stability, which may contain precipitated Hg salts depending on storage time and conditions. Further variability results from different degrees of bromination of the fluorescein backbone of the compound. The formation of mercury compound-protein adducts was detected. The peptide sequence T13 containing a free thiol residue was identified as the binding site for mercury species after tryptic digestion of beta-lactoglobulin A. While fresh Mercurochrome tends to the formation of a Hg(II)-beta-LGA adducts due to excess Hg(2+) in solution, investigations after precipitation of Hg salts yield Hg(merbromin)(beta-LGA) as the major product.

  12. Hydrophilic interaction liquid chromatography method development and validation for the assay of HEPES zwitterionic buffer.

    PubMed

    Xu, Xiaolong; Gevaert, Bert; Bracke, Nathalie; Yao, Han; Wynendaele, Evelien; De Spiegeleer, Bart

    2017-02-20

    HEPES is a zwitterionic buffer component used as a raw material in the GMP-manufacturing of advanced therapy medicinal products (ATMPs), hence requiring an adequate assay method with sufficient selectivity toward related impurities. Therefore, a hydrophilic interaction chromatography (HILIC) method was developed. Different factors were investigated towards the retention behavior of HEPES, its analogue EPPS and its starting material isethionate: pH, ion concentration and organic solvent ratio of the mobile phase, as well as column temperature. Moreover, stress testing resulted in the N-oxide degradant, identified by high resolution MS. The final method consisted of an isocratic system with an aqueous (pH 2.0 with H3PO4) acetonitrile (35:65, v/v) mobile phase on a zwitterionic HILIC (Obelisc N) column with a flow rate of 0.5mL/min and UV detection at 195nm. The assay method of HEPES was validated, obtaining adequate linearity (R(2)=0.999), precision (RSD of 0.5%) and accuracy (recovery of 100.08%). Finally, the applicability of the validated method was demonstrated by analysis of samples from different suppliers.

  13. Determination of iminosugars in mulberry leaves and silkworms using hydrophilic interaction chromatography-tandem mass spectrometry.

    PubMed

    Nakagawa, Kiyotaka; Ogawa, Kenta; Higuchi, Ohki; Kimura, Toshiyuki; Miyazawa, Teruo; Hori, Masatoshi

    2010-09-15

    Mulberry 1-deoxynojirimycin (DNJ, a potent alpha-glycosidase inhibitor) has been investigated thoroughly for its analytical methods and therapeutic potential against diabetes, whereas little attention has been given to other iminosugars such as 2-O-alpha-D-galactopyranosyl-DNJ (GAL-DNJ) and fagomine. For instance, concentration and composition of these iminosugars in mulberry leaves as well as sericulture products have not been fully characterized due to lack of suitable analytical methods. Here we developed a simultaneous determination method for DNJ, GAL-DNJ, and fagomine using hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS). When mulberry leaf extracts were subjected to HILIC-MS/MS with multiple reaction monitoring (MRM), individual iminosugars could be separated and detected. The developed method is sufficiently sensitive for determining iminosugars in mulberry leaves as well as silkworms, providing new information (e.g., different amounts of iminosugars in mulberry leaf varieties; high DNJ and low GAL-DNJ in the silkworm body, especially in the blood) that is useful for producing iminosugar-rich products for nutraceutical purposes. 2010 Elsevier Inc. All rights reserved.

  14. Effect of electric field on the partitioning behavior of solutes in entropic interaction chromatography.

    PubMed

    Shi, Qing-Hong; Jia, Guo-Dong; Xu, Liang; Sun, Yan

    2013-09-01

    In this study, a novel column design with a round cross-section was proposed to be suitable for a transverse electric field (EF). Additionally, two beads for entropic interaction chromatography (EIC) were prepared by grafting glycidyl methacrylate onto Toyopearl HW-65F (T65F) beads. Solute partitioning was then investigated to elucidate the role of graft polymerization with and without an EF. In a T65F column, solute partitioning was attributed to the distinct pore structure in the beads and was governed by pore flow. Under EF, partition coefficients (Kp) for solutes decreased with increasing EF strength. In the two EIC columns, a decrease of Kp was also observed without an EF while the fractionation windows were extended. It was more pronounced in the EIC column with a high grafting density (T65F-H). This was explained by the decrease in the effective pore size of solutes caused by the steric hindrance of polymer chains. Under an EF, the solutes showed different partitioning behaviours in the T65F-H column. With increasing EF strength, Kp for vitamin B12 and myoglobin was decreased. In contrast, Kp for large solutes increased as a result of concentration polarization on the bead surface. Both behaviors were related to the modulation of graft polymerization to residual charge on the matrix and the pore size of the solutes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Lipidomic profiling of dried seahorses by hydrophilic interaction chromatography coupled to mass spectrometry.

    PubMed

    Shen, Qing; Dai, Zhiyuan; Huang, Yao-Wen; Cheung, Hon-Yeung

    2016-08-15

    Dried seahorse is a precious raw food material for cooking soups. In this study, a lipidomics strategy using the techniques of solid-phase extraction (SPE) and hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-QTOF/MS) was developed for extraction, visualization, and quantification of phospholipids in dried seahorses. The parameters of SPE were optimized, and 1 mL of sample and chloroform/methanol (1:2, v/v) were found to be the best loading volume and eluting solvent, respectively. Afterwards, each phospholipid class was successfully separated on a HILIC column and analyzed by mass spectrometry. A total of 50 phospholipid molecular species were identified and determined, including 15 phosphatidylcholines (PCs), 14 phosphatidylethanolamines (PEs), 12 phosphatidylinositols (PIs) and 9 phosphatidylserines (PSs). In comparison to previously methods, this strategy was robust and efficient in extraction, characterization, and determination of phospholipids. The dried seahorse was found to contain large amounts of polyunsaturated fatty acyl phospholipids which are beneficial to human health. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Hydrophilic interaction liquid chromatography method for measuring the composition of aquatic humic substances.

    PubMed

    Wang, Ren-Qi; Gutierrez, Leonardo; Choon, Ng Siu; Croué, Jean-Philippe

    2015-01-01

    A hydrophilic interaction liquid chromatography (HILIC) method was developed to measure the composition of humic substances from river, reservoir, and treated wastewater based on their physicochemical properties. The current method fractionates the humic substances into four well-defined groups based on parallel analyses with a neutral and a cationic HILIC column, using mobile phases of varied compositions and pH. The results indicate that: (i) the proportion of carboxylic acids in the humic substances from terrestrial origins is less than half of that from treated wastewater (Jeddah, KSA), (ii) a higher content of basic compounds was observed in the humic substances from treated wastewater and Ribou Reservoir (Cholet, France) than in the sample from Loire River (France), (iii) a higher percentage of hydrophobic macromolecules were found in the humic substances from Loire River than in the other samples, and (iv) humic substances of treated wastewater contained less ionic neutral compounds (i.e., pKa 5-9) than the waters from terrestrial origins. The physicochemical property disparity amongst the compounds in each humic substances sample was also evaluated. The humic substances from the lightly humic Loire river displayed the highest disparity, whereas the highly humic Suwannee river (Georgia, USA) showed the most homogeneous humic substances. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Molecular approach to protein-polymer interactions in ion-exchange chromatography.

    PubMed

    Noinville, V; Craescu, C T; Vidal-Madjar, C; Sebille, B

    1995-02-03

    A model was developed and implemented to aid in understanding and predicting the retention behaviour of proteins in ion-exchange chromatography. The model structures chosen were calcium-loaded and -depleted alpha-lactalbumin (ALC) and hen egg white lysozyme (HEWL) and a comparison was made with chromatographic measurements. A characteristic charge of -3.4 was found under the experimental conditions applied for both forms of ALC, and HEWL was not retained. The model explicitly considers all of the atoms, each being assigned a set of force field parameters. Because of the computational time necessary to include them, water molecules were not taken into account, but a sigmoidal function of the dielectric permittivity was introduced in the calculations. Interaction potential energies from bulk down to the contact were evaluated for each protein. The results were in qualitative agreement with those of the chromatographic experiments. It was possible to reproduce the difference in retention between both forms of ALC and also the behaviour of HEWL.

  18. Quantification of plasma homocitrulline using hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry.

    PubMed

    Jaisson, Stéphane; Gorisse, Laëtitia; Pietrement, Christine; Gillery, Philippe

    2012-02-01

    Homocitrulline (HCit), an amino acid formed by the carbamylation of ε-amino groups of lysine residues, is considered a promising biomarker for monitoring diseases such as chronic renal failure and atherosclerosis. This paper describes a tandem mass spectrometric method for total, protein-bound and free HCit measurement in plasma samples. HCit was separated from other plasma components by hydrophilic interaction liquid chromatography. Detection was achieved by monitoring transitions of 190.1 > 127.1 and 190.1 > 173.1 for HCit, and 183.1 > 120.2 for d(7)-citrulline used as internal standard. This method allowed HCit quantification within 5.2 min and was precise (inter-assay CV < 5.85%), accurate (mean recoveries ranging from 97% to 106%), and exhibited a good linearity from 10 nmol/L to 1.6 μmol/L. Plasma samples from control and uremic mice (n = 10) were analyzed. In control mice, mean total plasma HCit concentration was 0.78 ± 0.12 μmol/mol amino acids, whereas it was increased 2.7-fold in uremic mice plasma, reaching 2.10 ± 0.50 μmol/mol amino acids (p < 0.001). In conclusion, this method exhibits good analytical performances and meets the criteria of sensitivity suitable for HCit concentration assessment in plasma samples.

  19. Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments.

    PubMed

    Yeh, Chia-Hao; Chen, Shu-Hui; Li, Ding-Tzai; Lin, Hong-Ping; Huang, Hung-Jen; Chang, Chi-I; Shih, Wen-Ling; Chern, Chi-Liang; Shi, Fong-Ku; Hsu, Jue-Liang

    2012-02-10

    Purification of glycopeptides prior to the analysis by mass spectrometry (MS) is demanded due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among various purification methods, hydrophilic interaction liquid chromatography (HILIC) has become a popular method in recent years. In this work, we reported a novel magnetic bead-based zwitterionic HILIC (ZIC-HILIC) material which was fabricated by coating a zwitterionic polymer synthesized by spontaneous acid-catalyzed polymerization of 4-vinyl-pyridinium ethanesulfonate monomer on iron oxide magnetic nanoparticles. The resulting magnetic ZIC-HILIC nanoparticles were shown to provide high specificity and high recovery yield (95-100%) for the enrichment of glycopeptides from a standard glycoprotein, fetuin, using a simple magnetic bar. In addition, we proposed a two-step HILIC enrichment strategy using magnetic ZIC-HILIC nanoparticles for a large scale analysis of glycoproteins in complex biological samples. Using this approach, we identified 85 N-glycosylation sites in 53 glycoproteins from urine samples. Two novel glycosylation sites on N513 of uromodulin and N470 of lysosomal alpha-glucosidase which have not yet been reported were identified by two-step HILIC approach. Furthermore, all these identified sites were confirmed by studies conducted using PNGase F deglycosylation and 18O enzymatic labeling. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions.

    PubMed

    Zacharis, Constantinos K; Kalaitzantonakis, Eftichios A; Podgornik, Ales; Theodoridis, Georgios

    2007-03-09

    In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).

  1. Time scale of stationary decoherence

    NASA Astrophysics Data System (ADS)

    Polonyi, Janos

    2017-07-01

    The decoherence of a test particle interacting with an ideal gas is studied by the help of the effective Lagrangian, derived in the leading order of the perturbation expansion and in order O (∂t2) . The stationary decoherence time is found to be comparable to or longer than the diffusion time. The decoherence time reaches its minimal value for classical, completely decohered environment, suggesting that physical decoherence is slowed down as compared with diffusion by the quantum coherence of the environment.

  2. Rapid determination of glyphosate, glufosinate, bialaphos, and their major metabolites in serum by liquid chromatography-tandem mass spectrometry using hydrophilic interaction chromatography.

    PubMed

    Yoshioka, Naoki; Asano, Migiwa; Kuse, Azumi; Mitsuhashi, Takao; Nagasaki, Yasushi; Ueno, Yasuhiro

    2011-06-10

    We developed a simple and rapid method for the simultaneous determination of phosphorus-containing amino acid herbicides (glyphosate, glufosinate, bialaphos) and their major metabolites, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicopropionic acid (MPPA), in human serum. Serum samples were filtrated through an ultrafiltration membrane to remove proteins. The filtrate was then washed with chloroform, and injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Chromatographic separation was achieved on a hydrophilic interaction chromatography (HILIC) column. Determination of the target herbicides and metabolites was successfully carried out without derivatization or solid phase extraction (SPE) cartridge clean-up. The recoveries of these compounds, added to human serum at 0.2μg/mL, ranged from 94% to 108%, and the relative standard deviations (RSDs) were within 5.9%. The limits of detection (LODs) were 0.01μg/mL for MPPA, 0.02μg/mL for AMPA, 0.03μg/mL for both glyphosate and glufosinate, and 0.07μg/mL for bialaphos, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Impact of organic modifier and temperature on protein denaturation in hydrophobic interaction chromatography.

    PubMed

    Bobaly, Balázs; Beck, Alain; Veuthey, Jean-Luc; Guillarme, Davy; Fekete, Szabolcs

    2016-11-30

    The goal of this study was to better understand the chromatographic conditions in which monoclonal antibodies (mAbs) of broad hydrophobicity scale and a cysteine conjugated antibody-drug conjugate (ADCs), namely brentuximab-vedotin, could denaturate. For this purpose, some experiments were carried out in HIC conditions using various organic modifier in natures and proportions, different mobile phase temperatures and also different pHs. Indeed, improper analytical conditions in hydrophobic interaction chromatography (HIC) may create reversed-phase (RP) like harsh conditions and therefore protein denaturation. In terms of organic solvents, acetonitrile (ACN) and isopropanol (IPA) were tested with proportions ranging from 0 to 40%. It appeared that IPA was a less denaturating solvent than ACN, but should be used in a reasonable range (10-15%). Temperature should also be kept reasonable (below 40°C), to limit denaturation under HIC conditions. However, the combined increase of temperature and organic content induced denaturation of protein biopharmaceuticals in all cases. Indeed, above 30-40°C and 10-15% organic modifier in mobile phase B, heavy chain (HC) and light chain (LC) fragments dissociated. Mobile phase pH was also particularly critical and denaturation was significant even under moderately acidic conditions (pH of 5.4). Today, HIC is widely used for measuring drug-to-antibody ratio (DAR) of ADCs, which is a critical quality attribute of such samples. Here, we demonstrated that the estimation of average DAR can be dependent on the amount of organic modifier in the mobile phase under HIC conditions, due to the better recovery of the most hydrophobic proteins in presence of organic solvent (IPA). So, special care should be taken when measuring the average DAR of ADCs in HIC. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Identification and quantification of nucleosides and nucleobases in Geosaurus and Leech by hydrophilic-interaction chromatography.

    PubMed

    Chen, Pei; Li, Wei; Li, Qin; Wang, Yinghua; Li, Zhenguo; Ni, Yefeng; Koike, Kazuo

    2011-09-15

    A simple hydrophilic-interaction chromatography (HILIC) method was developed for the identification and quantification of 14 nucleosides and nucleobases, namely cytosine, uracil, cytidine, guanine, hypoxanthine, xanthine, uridine, thymine, inosine, guanosine, thymidine, 2'-deoxyadenosine, 2'-deoxyinosine and 2'-deoxyuridine in two traditional Chinese medicines, Geosaurus and Leech. The separation was achieved on a TSKgel Amide-80 column (150 mm × 2.0 mm, 3.0 μm) with a mixture of acetonitrile and 10 mM aqueous ammonium acetate as the mobile phase at a flow rate of 0.2 mL/min. The temperature was set at 30°C and UV detection wavelength was set at 260 nm. All calibration curves showed good linearity (R(2)>0.9957) within the test ranges. The overall intra- and inter-day RSD ranged from 0.4 to 3.4% and from 0.7 to 3.3%, respectively. The LOD and LOQ were in the range of 0.07-30.49 ng/mL and 0.26-60.98 ng/mL, respectively. The repeatability of the method was in the range of 2.2-5.8% for Geosaurus and 1.4-5.5% for Leech. The recoveries of the samples were in the range of 91.4-100.9% for Geosaurus, and 91.9-99.3% for Leech. The established method was applied successfully for the analysis of nucleosides and nucleobases in 22 commercially available samples collected from different regions in China and Japan. Our data showed that HILIC had advantages as a useful tool for the study of the bioactive components in Geosaurus and Leech as well as their quality control, and could therefore be used for the determination of the analytes in pharmaceutical products and biological fluids.

  5. Thermodynamic modelling of hydrophobic interaction chromatography of biomolecules in the presence of salt.

    PubMed

    Mirani, Mohammad Reza; Rahimpour, Farshad

    2015-11-27

    Hydrophobic interaction chromatography (HIC) is a useful method for isolation and purification of macromolecules. HIC separates proteins on the basis of surface hydrophobicity while generally retaining the activity of proteins. Aqueous mobile phases with high salt concentrations are often used to adsorb the proteins on a mildly hydrophobic support. In this research, the thermodynamic model of Chen and Sun, which predicts the adsorption isotherms of protein in presence of different type of salts, was modified by substitution the protein and salt activities in the mobile phase instead of their concentrations. In addition, model was examined for studying the adsorption of BSA, HSA, α-lactalbumin and Trypsinogen on different sepharose gels. The model parameters of Chen and Sun are adsorption equilibrium constant (KP), protein dehydration equilibrium constant (Ks), salt coefficient (α) and number of ligand binding (n). By substitution activity instead of salt and protein concentration, two other parameters (c1 and As), which related to the activity coefficients, are added to the model. The parameters of this nonlinear model are calculated by genetic algorithm (GA). The maximum average absolute percentage deviation (AAD) for the data which are obtained from the adsorption isotherm of BSA on phenyl sepharose gel, in the presence of different concentration of NaCl was 4.8%, while for Chen and Sun model, was 22.0%. Also maximum ADD for HSA, α-lactalbumin, and Trypsinogen adsorption was 7.8, 6.9, and 8.4, respectively. The results indicate that the modified model has adequate accuracy to predict protein HIC behaviour.

  6. Prediction of retention times of proteins in hydrophobic interaction chromatography using only their amino acid composition.

    PubMed

    Salgado, J Cristian; Rapaport, Ivan; Asenjo, Juan A

    2005-12-09

    This paper focuses on the prediction of the dimensionless retention time of proteins (DRT) in hydrophobic interaction chromatography (HIC) by means of mathematical models based, essentially, only on aminoacidic composition. The results show that such prediction is indeed possible. Our main contribution was the design of models that predict the DRT using the minimal information concerning a protein: its aminoacidic composition. The performance is similar to that observed in models that use much more sophisticated information such as the three-dimensional structure of proteins. Three models that, in addition to the amino acid composition, use different assumptions about the amino acids tendency to be exposed to the solvent, were evaluated in 12 proteins with known experimental DRT. In all the cases analyzed, the model that obtained the best results was the one based on a linear estimation of the aminoacidic surface composition. The models were adjusted using a collection of 74 vectors of aminoacidic properties plus a set of 6388 vectors derived from these using two mathematical tools: k-means and self-organizing maps (SOM) algorithms. The best vector was generated by the SOM algorithm and was interpreted as a hydrophobicity scale based partly on the tendency of the amino acids to be hidden in proteins. The prediction error (MSE(JK)) obtained by this model was almost 35% smaller than that obtained by the model that supposes that all the amino acids are completely exposed and 40% smaller than that obtained by the model that uses a simple correction factor considering the general tendency of each amino acid to be exposed to the solvent. In fact, the performance of the best model based on the aminoacidic composition was 5% better than that observed in the model based on the three-dimensional structure of proteins.

  7. Hydrophilic interaction liquid chromatography for the separation, purification, and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz.

    PubMed

    Liang, Tu; Fu, Qing; Li, Fangbing; Zhou, Wei; Xin, Huaxia; Wang, Hui; Jin, Yu; Liang, Xinmiao

    2015-08-01

    A systematic strategy based on hydrophilic interaction liquid chromatography was developed for the separation, purification and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz. Methods with enough hydrophilicity and selectivity were utilized to resolve the problems encountered in the separation of oligosaccharides such as low retention, low resolution and poor solubility. The raffinose family oligosaccharides in L. lucidus Turcz. were isolated using solid-phase extraction followed by hydrophilic interaction liquid chromatography at semi-preparative scale to obtain standards of stachyose, verbascose and ajugose. Utilizing the obtained oligosaccharides as standards, a quantitative determination method was developed, validated and applied for the content determination of raffinose family oligosaccharides both in the aerial and root parts of L. lucidus Turcz. There were no oligosaccharides in the aerial parts, while in the root parts, the total content was 686.5 mg/g with the average distribution: raffinose 66.5 mg/g, stachyose 289.0 mg/g, verbascose 212.4 mg/g, and ajugose 118.6 mg/g. The result provided the potential of roots of L. lucidus Turcz. as new raffinose family oligosaccharides sources for functional food. Moreover, since the present systematic strategy is efficient, sensitive and robust, separation, purification and quantification of oligosaccharides by hydrophilic interaction liquid chromatography seems to be possible.

  8. Mixed-mode ion-exchangers and their comparative chromatographic characterization in reversed-phase and hydrophilic interaction chromatography elution modes.

    PubMed

    Lämmerhofer, Michael; Richter, Martin; Wu, Junyan; Nogueira, Raquel; Bicker, Wolfgang; Lindner, Wolfgang

    2008-08-01

    A set of particulate silica-supported mixed-mode RP/weak anion-exchangers (RP/WAX) (obtained by bonding of N-undecenoylated 3-aminoquinuclidine, 3-aminotropane and 2-dimethylaminoethylamine as well as of N-butenoyl-(2S,4S,5R)-2-aminomethyl-5-[(2-octylthio)ethyl]-quinuclidine to thiol-modified silica) were chromatographically characterized in comparison to selected commercially available columns using two distinct isocratic elution modes, viz. an aqueous-rich RP-type elution mode (with 40% ACN and 60% buffer) as well as an organic solvent-rich hydrophilic interaction chromatography (HILIC)-type elution mode (95 and 90% ACN). The mixed-mode RP/WAX phases showed multimodal applicability, unlike a polar embedded RP material (Synergi Fusion RP), amino phases (Luna NH(2), BioBasic AX) or typical HILIC packings (ZIC-HILIC, TSKGel Amide-80). Principal component analysis (PCA) of the RP test data confirmed that the in-house developed RP/WAX columns as well as the Acclaim Mixed-Mode WAX-1 phase resemble each other in their chromatographic characteristics having slightly lower hydrophobic selectivity (alpha(CH2) of 1.5) than the tested Synergi Fusion RP (alpha(CH2) approximately 1.8). In contrast, a decrease in mixed-mode character due to lowered ion-exchange capacity and concomitantly increased RP-like behavior could be identified for other mixed-mode phases in the order of Obelisc R > Primesep B2 > Uptisphere MM3. PCA on HILIC data revealed that the RP/WAX phases behave dissimilar to TSKGel Amide-80, ZIC-HILIC and polysulfoethyl A under the chosen elution conditions. Hence, they may be regarded as complementary to these commercial stationary phases with applicability profiles for hydrophilic but also hydrophobic solutes.

  9. Development and validation of a hydrophilic interaction chromatography method coupled with a charged aerosol detector for quantitative analysis of nonchromophoric α-hydroxyamines, organic impurities of metoprolol.

    PubMed

    Xu, Qun; Tan, Shane; Petrova, Katya

    2016-01-25

    The European Pharmacopeia (EP) metoprolol impurities M and N are polar, nonchromophoric α-hydroxyamines, which are poorly retained in a conventional reversed-phase chromatographic system and are invisible for UV detection. Impurities M and N are currently analyzed by TLC methods in the EP as specified impurities and in the United States Pharmacopeia-National Formulary (USP-NF) as unspecified impurities. In order to modernize the USP monographs of metoprolol drug substances and related drug products, a hydrophilic interaction chromatography (HILIC) method coupled with a charged aerosol detector (CAD) was explored for the analysis of the two impurities. A comprehensive column screening that covers a variety of HILIC stationary phases (underivatized silica, amide, diol, amino, zwitterionic, polysuccinimide, cyclodextrin, and mixed-mode) and optimization of HPLC conditions led to the identification of a Halo Penta HILIC column (4.6 × 150 mm, 5 μm) and a mobile phase comprising 85% acetonitrile and 15% ammonium formate buffer (100 mM, pH 3.2). Efficient separations of metoprolol, succinic acid, and EP metoprolol impurities M and N were achieved within a short time frame (<8 min). The HILIC-CAD method was subsequently validated per USP validation guidelines with respect to specificity, robustness, linearity, accuracy, and precision, and could be incorporated into the current USP-NF monographs to replace the outdated TLC methods. Furthermore, the developed method was successfully applied to determine organic impurities in metoprolol drug substance (metoprolol succinate) and drug products (metoprolol tartrate injection and metoprolol succinate extended release tablets).

  10. Isolation of fluorescent constituents from soil humic and fulvic acids by hydrophilic interaction chromatography

    NASA Astrophysics Data System (ADS)

    Aoyama, Masakazu

    2014-05-01

    Humic acids (HAs) and fulvic acids (FAs) are the most abundant components of soil organic matter and exhibit fluorescence. Our previous studies using high performance size-exclusion chromatography (HPSEC) and polyacrylamide gel electrophoresis demonstrated that the fluorescence of soil HAs was mainly due to the minor constituents with relatively small molecular sizes. In order to clarify the nature of the fluorescence of soil organic matter, it is necessary to isolate the fluorescent constituents from HAs and FAs. I succeeded in isolating the fluorescent constituents from soil HAs and FAs by using hydrophilic interaction chromatography (HILIC). When HILIC of soil HAs and FAs was carried out under isocratic conditions using a SeQuant ZIC-HILIC column and acetonitrile-water as a mobile phase, the complete separation of fluorescent and non-fluorescent peaks was achieved at the acetonitrile concentration of 90%. Another fluorescent peak was eluted with decreasing concentration of acetonitrile from 90% to 50%. The use of a TSKgel Amide-80 column gave the same results. The best resolution was obtained when HILIC was performed under gradient conditions from 90% to 50% acetonitrile using the ZIC-HILIC and Amide-80 columns linked in series. For both HAs and FAs, a sharp non-fluorescent peak (peak A) followed by a sharp fluorescent peak (peak B) and a broad fluorescent peak (peak C) were eluted under the above optimum operating conditions. The intensity of peak A relative to that of peak B was significantly less in the FAs than in the HAs. The fluorescent peaks (peaks B and C) of the FAs showed considerable UV absorption, whereas those of the HAs did little UV absorption. When the fluorescence emission spectra (excitation at 280 nm) were measured for the fluorescent peaks, two emission peaks were located at 460 and 520 nm for the HAs, while for the FAs, a broad emission peak at 400-450 nm with a small shoulder at around 500 nm was observed. The peaks were collected

  11. A simultaneous determination method for 5-fluorouracil and its metabolites in human plasma with linear range adjusted by in-source collision-induced dissociation using hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry.

    PubMed

    Ishii, Hideaki; Shimada, Miki; Yamaguchi, Hiroaki; Mano, Nariyasu

    2016-11-01

    We applied a new technique for quantitative linear range shift using in-source collision-induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5-fluorouracil (5-FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). To control adverse effects after administration of 5-FU, it is important to monitor the plasma concentration of 5-FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5-FU and its metabolites in human plasma by LC/ESI-MS/MS coupled with the technique for quantitative linear range shift using in-source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5-FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile-rich eluent after LC separation improved the ESI-MS response of high polar analytes. Based on the validation results, linear range shifts by in-source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  12. Analysis of phospholipids in bio-oils and fats by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Viidanoja, Jyrki

    2015-09-15

    A new, sensitive and selective liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method was developed for the analysis of Phospholipids (PLs) in bio-oils and fats. This analysis employs hydrophilic interaction liquid chromatography-scheduled multiple reaction monitoring (HILIC-sMRM) with a ZIC-cHILIC column. Eight PL class selective internal standards (homologs) were used for the semi-quantification of 14 PL classes for the first time. More than 400 scheduled MRMs were used for the measurement of PLs with a run time of 34min. The method's performance was evaluated for vegetable oil, animal fat and algae oil. The averaged within-run precision and between-run precision were ≤10% for all of the PL classes that had a direct homologue as an internal standard. The method accuracy was generally within 80-120% for the tested PL analytes in all three sample matrices.

  13. Comprehensive hydrophilic interaction and ion-pair reversed-phase liquid chromatography for analysis of di- to deca-oligonucleotides.

    PubMed

    Li, Qin; Lynen, Frédéric; Wang, Jian; Li, Hanlin; Xu, Guowang; Sandra, Pat

    2012-09-14

    A comprehensive two-dimensional HPLC approach with a high degree of orthogonality was developed for analysis of di- to deca-oligonucleotides (ONs). Hydrophilic interaction liquid chromatography (HILIC) was used in the first dimension, and ion-pair reversed-phase liquid chromatography (IP-RPLC) was employed in the second dimension. The two dimensions were connected via a ten-port valve interface equipped with octadecyl silica (ODS) traps to immobilize and focus the ONs eluting from the first dimension prior to IP-RPLC separation. An aqueous make-up flow was used for effective trapping. The comprehensive two-dimensional HPLC system was optimized with a mixture consisting of 27 oligonucleotide standards. An overall chromatographic peak capacity of 500 was obtained. The use of the volatile buffer triethylamine acetate in the second dimension allowed straightforward coupling to electrospray ionization mass spectrometry (ESI-MS) and detection of each ON in the negative ionization mode.

  14. Recent advances in hydrophilic interaction chromatography for quantitative analysis of endogenous and pharmaceutical compounds in plasma samples.

    PubMed

    Isokawa, Muneki; Kanamori, Takahiro; Funatsu, Takashi; Tsunoda, Makoto

    2014-09-01

    There is an increasing need for new analytical methods that can handle a large number of analytes in complex matrices. Hydrophilic interaction chromatography (HILIC) has recently been demonstrated as an important supplement to reversed-phase liquid chromatography for polar analytes, particularly endogenous compounds. With the increasing popularity of HILIC, progressively more polar phases with diverse functional groups have been developed. In addition, the coupling of HILIC to mass spectrometry offers the advantages of improved sensitivity by employing an organic-rich mobile phase. This article reviews recent applications of HILIC for the analysis of endogenous and pharmaceutical compounds in plasma samples. Furthermore, based on recent studies, we provide a discussion of column selection, sample pretreatment for HILIC analysis, and detection sensitivity.

  15. [Determination of melamine and ammeline in eggs and meat using hydrophilic interaction liquid chromatography].

    PubMed

    Li, Yanzhao; Hao, Weiqiang; Wang, Yubo; Chen, Qiang; Li, Jinchun; Sun, Xiaoli

    2012-07-01

    A hydrophilic interaction liquid chromatographic (HILIC) method for the determination of melamine and its degradation product ammeline in eggs and meat has been developed. The separation was carried out on a ZIC-HILIC column with 3 mmol/L NH4H2PO4 (pH 6.9)-acetonitrile (20: 80, v/v) as mobile phase at the flow rate of 0.8 mL/min, and detected at 220 nm. Compared with the reversed-phase liquid chromatography, this method can avoid the use of ion pair reagents and thus simplify the composition of mobile phase. Under the above chromatographic conditions, melamine and ammeline had good peak shapes and moderate retention times. Good separation between these compounds and the substances that were naturally contained in the samples can be achieved. For the sample preparation, the analytes were first extracted with 0.1% phosphoric acid due to the basicity of melamine and ammeline. Then, metaphosphoric acid and acetonitrile were used to remove proteins and saccharides by precipitation. After the filtration and removal of acetonitrile by rotary evaporation under vacuum, the filtrate was cleaned-up by solid-phase extraction (SPE) technique in which a cation exchange column was used. The SPE column was activated by using methanol and 0.1% phosphoric acid. A solution of 5% ammonia methanol was chosen as eluent. The residues obtained from the eluant by evaporating the solvent were resolved in the mobile phase. It was found that there was a good linear relationship between concentration and detector response within the range of 0.4-40 mg/L. The limits of detection were 2 mg/kg for both melamine and ammeline. The average recoveries were between 80% and 105% in the spiked range of 2-10 mg/kg. The relative standard deviations were not more than 10%. The solutions of melamine and ammeline were stable in a month. The established method can be used in practice to determine melamine and ammeline simultaneously in egg and meat samples.

  16. Development of an improved online comprehensive hydrophilic interaction chromatography × reversed-phase ultra-high-pressure liquid chromatography platform for complex multiclass polyphenolic sample analysis.

    PubMed

    Sommella, Eduardo; Ismail, Omar H; Pagano, Francesco; Pepe, Giacomo; Ostacolo, Carmine; Mazzoccanti, Giulia; Russo, Mariateresa; Novellino, Ettore; Gasparrini, Francesco; Campiglia, Pietro

    2017-05-01

    In this study, an improved online comprehensive two-dimensional liquid chromatography platform coupled to tandem mass spectrometry was developed for the analysis of complex polyphenolic samples. A narrowbore hydrophilic interaction chromatography column (150 × 2.0 mm, 3.0 μm, cross-linked diol) was employed in the first dimension, while a reversed-phase column based on monodisperse sub-2 μm fully porous particles (50 × 3.0 mm, 1.9 μm d.p.) with high surface area (410 m(2) /g) was employed in the second dimension. The combination of a trapping column modulation interface with the high retentive fully porous monodisperse reversed-phase column in the second dimension resulted in higher peak capacity values (1146 versus 867), increased sensitivity, sharper and more symmetrical peaks in comparison with a conventional loop-based method, with the same analysis time (70 min). The system was challenged against a complex polyphenolic extract of a typical Italian apple cultivar, enabling the simultaneous separation of multiple polyphenolic classes, including oligomeric procyanidins, up to degree of polymerization of 10. Hyphenation with an ion trap time-of-flight mass spectrometer led to the tentative identification of 121 analytes, showing how this platform could be a powerful analytical tool for the accurate profiling of complex polyphenolic samples. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. An Advanced, Interactive, High-Performance Liquid Chromatography Simulator and Instructor Resources

    ERIC Educational Resources Information Center

    Boswell, Paul G.; Stoll, Dwight R.; Carr, Peter W.; Nagel, Megan L.; Vitha, Mark F.; Mabbott, Gary A.

    2013-01-01

    High-performance liquid chromatography (HPLC) simulation software has long been recognized as an effective educational tool, yet many of the existing HPLC simulators are either too expensive, outdated, or lack many important features necessary to make them widely useful for educational purposes. Here, a free, open-source HPLC simulator is…

  18. An Advanced, Interactive, High-Performance Liquid Chromatography Simulator and Instructor Resources

    ERIC Educational Resources Information Center

    Boswell, Paul G.; Stoll, Dwight R.; Carr, Peter W.; Nagel, Megan L.; Vitha, Mark F.; Mabbott, Gary A.

    2013-01-01

    High-performance liquid chromatography (HPLC) simulation software has long been recognized as an effective educational tool, yet many of the existing HPLC simulators are either too expensive, outdated, or lack many important features necessary to make them widely useful for educational purposes. Here, a free, open-source HPLC simulator is…

  19. Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals.

    PubMed

    Periat, Aurélie; Fekete, Szabolcs; Cusumano, Alessandra; Veuthey, Jean-Luc; Beck, Alain; Lauber, Matthew; Guillarme, Davy

    2016-05-27

    A new stationary phase based on wide-pore hybrid silica bonded with amide ligand has been used to explore the utility of HILIC for the analytical characterization of protein biopharmaceuticals. Various, highly-relevant samples were tested, including different insulins, interferon α-2b and trastuzumab. This work shows that HILIC can be successfully employed for the analysis of therapeutic proteins and mAbs, using mobile phase compositions comprised of between 65 and 80% ACN and 0.1% TFA. In terms of elution order and selectivity, these HILIC separations have proven to be highly orthogonal to RPLC, while the kinetic performance remains comparable. In the case of characterizing trastuzumab, HILIC was uniquely able to resolve several important glycoforms at the middle-up level of analysis (fragments of 25-100kDa). Such a separation of glycoforms has been elusive by other separation mechanisms, such as RPLC and IEX. Besides showing orthogonality to RPLC and improved separations of glycoforms, HILIC offers several additional benefits for biopharmaceutical characterization: i) an inherent compatibility with MS, ii) a reduced requirement for very high mobile phase temperatures that are otherwise needed in RPLC to limit undesirably strong adsorption to the surface of the stationary phase, and iii) the possibility to couple several columns in series to improve resolving power, thanks to comparatively low mobile phase viscosity.

  20. Hydrophilic interaction liquid chromatography-solid phase extraction directly combined with protein precipitation for the determination of triptorelin in plasma.

    PubMed

    Wang, Jixia; Kong, Song; Yan, Jingyu; Jin, Gaowa; Guo, Zhimou; Shen, Aijin; Xu, Junyan; Zhang, Xiuli; Zou, Lijuan; Liang, Xinmiao

    2014-06-01

    Peptide drugs play a critical role in therapeutic treatment. However, as the complexity of plasma, determination of peptide drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a daunting task. To solve this problem, hydrophilic interaction liquid chromatography-solid phase extraction (HILIC-SPE) directly combined with protein precipitation (PPT) was developed for the selective extraction of triptorelin from plasma. The extracts were analyzed by reversed-phase liquid chromatography (RPLC). Proteins, phospholipids and highly polar interferences could be removed from plasma by the efficient combination of PPT, HILIC-SPE and RPLC-MS/MS. This method was evaluated by matrix effect, recovery and process efficiency at different concentration levels (50, 500 and 5,000 ng/mL) of triptorelin. Furthermore, the performance of HILIC-SPE was compared with that of reversed-phase C18 SPE and hydrophilic lipophilic balance (Oasis HLB) SPE. Among them, HILIC-SPE provided the minimum matrix effect (ranging from 96.02% to 103.41%), the maximum recovery (ranging from 80.68% to 90.54%) and the satisfactory process efficiency (ranging from 82.83% to 92.95%). The validated method was successfully applied to determine triptorelin in rat plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for acidic herbicides and metabolites analysis in fresh water.

    PubMed

    Fauvelle, Vincent; Mazzella, Nicolas; Morin, Soizic; Moreira, Sylvia; Delest, Brigitte; Budzinski, Hélène

    2015-03-01

    Theoretical papers and environmental applications of hydrophilic interaction liquid chromatography (HILIC) have been published for a wide range of analytes, but to our knowledge, no study focused on acidic herbicides (e.g., triketones, phenoxy aci