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Sample records for affinity column chromatography

  1. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  2. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario.

  3. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario. PMID:25748537

  4. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-01

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

  5. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  6. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies.

    PubMed

    Boulet-Audet, Maxime; Kazarian, Sergei G; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  7. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  8. Selective isolation of β-glucan from corn pericarp hemicelluloses by affinity chromatography on cellulose column.

    PubMed

    Yoshida, Tomoki; Honda, Yoichi; Tsujimoto, Takashi; Uyama, Hiroshi; Azuma, Jun-ichi

    2014-10-13

    A combination of anion-exchange chromatography and affinity chromatography on a cellulose column was found to be effective for the isolation of β-(1,3;1,4)-glucan (BG) from corn pericarp hemicelluloses (CPHs). CPHs containing 6.6% BG were extracted from corn pericarp with 6M urea-2 wt% NaOH solution and initially fractionated into neutral and acidic parts by anion exchange chromatography to remove acidic arabinoxylan consisting of arabinose (35.6%) and xylose (50.9%). The neutral fraction (yield; 10.1% on the basis of CPHs) consisting of 1.0% arabinose, 10.1% xylose and 80.3% glucose containing 28.4% BG was then applied to a cellulose column of Whatman CF-11. BG could be recovered from the adsorbed fraction on the cellulose column by elution with 2% NaOH in a yield of 2.6% on the basis of CPHs with a purity of 84.7%. The chemical structure of the isolated corn pericarp BG was confirmed by (13)C NMR spectroscopic, methylation and lichenase treatment analyses. The results indicate that the ratios of (1,4)/(1,3) linkage and cellotriosyl/cellotetraosyl segments of the BG were 2.60 and 2.5, respectively.

  9. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  10. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

    PubMed

    Habicht, K-L; Singh, N S; Indig, F E; Wainer, I W; Moaddel, R; Shimmo, R

    2015-09-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  11. Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

    PubMed Central

    Ruprecht, Benjamin; Koch, Heiner; Medard, Guillaume; Mundt, Max; Kuster, Bernhard; Lemeer, Simone

    2015-01-01

    Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO2, Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO2 in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems. PMID

  12. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    ERIC Educational Resources Information Center

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  13. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  14. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  15. Synthesis and characterization of a cellular membrane affinity chromatography column containing histamine 1 and P2Y1 receptors: A multiple G-protein coupled receptor column

    PubMed Central

    Moaddel, Ruin; Musyimi, Harrison K.; Sanghvi, Mitesh; Bashore, Charlene; Frazier, Chester R.; Khadeer, Mohammad; Bhatia, Prateek; Wainer, Irving W.

    2015-01-01

    A cellular membrane affinity chromatography (CMAC) column has been created using cellular membrane fragments from a 1321N1 cell line stably transfected with the P2Y1 receptor. The CMAC(1321N1P2Y1) column contained functional P2Y1 and histamine 1 receptors, which independently bound receptor-specific ligands. The data obtained with the CMAC(1321N1P2Y1) column demonstrate that multiple-G-protein coupled receptor (GPCR) columns can be developed and used to probe interactions with the immobilized receptors and that endogenously expressed GPCRs can be used to create CMAC columns. The results also establish that the histamine 1 receptor can be immobilized with retention of ligand-specific binding. PMID:19608372

  16. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 1: Theory

    PubMed Central

    2015-01-01

    We present a novel technique that couples isotachophoresis (ITP) with affinity chromatography (AC) to achieve rapid, selective purification with high column utilization. ITP simultaneously preconcentrates an analyte and purifies it, based on differences in mobility of sample components, excluding species that may foul or compete with the target at the affinity substrate. ITP preconcentration accelerates the affinity reaction, reducing assay time, improving column utilization, and allowing for capture of targets with higher dissociation constants. Furthermore, ITP-AC separates the target and contaminants into nondiffusing zones, thus achieving high resolution in a short distance and time. We present an analytical model for spatiotemporal dynamics of ITP-AC. We identify and explore the effect of key process parameters, including target distribution width and height, ITP zone velocity, forward and reverse reaction constants, and probe concentration on necessary affinity region length, assay time, and capture efficiency. Our analytical approach shows collapse of these variables to three nondimensional parameters. The analysis yields simple analytical relations for capture length and capture time in relevant ITP-AC regimes, and it demonstrates how ITP greatly reduces assay time and improves column utilization. In the second part of this two-part series, we will present experimental validation of our model and demonstrate ITP-AC separation of the target from 10,000-fold more-abundant contaminants. PMID:24937679

  17. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  18. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  19. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

  20. Characterization of a multiple endogenously expressed Adenosine triphosphate-Binding Cassette transporters using nuclear and cellular membrane affinity chromatography columns

    PubMed Central

    Khadeer, M.A.; Shimmo, R.; Wainer, I.W.; Moaddel, R.

    2014-01-01

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of 8 compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN229) column and decreased it (−5%) on the NMAC(LN229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

  1. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 2: Experimental Study

    PubMed Central

    2015-01-01

    We present an experimental study of coupling of isotachophoresis (ITP) and affinity chromatography (AC) to effect rapid, selective purification with high column utilization and high resolution. We provide a detailed protocol for performing ITP-AC and describe the design of a buffer system to perform sequence specific separation of nucleic acids. We describe the synthesis and functionalization of our affinity substrate, poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM). This substrate allows easy immobilization of affinity probes, is nonsieving (even to macromolecules), and exhibits negligible nonspecific binding. We demonstrate ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA probe and study the spatiotemporal dynamics using epifluorescence imaging. We make qualitative and quantitative comparisons between these data and the model presented in the first part of this two-paper series. We vary the target concentration from 1 pg μL–1 to 100 pg μL–1 and ITP velocity over the range of 10–50 μm s–1, and thereby explore over 4 orders of magnitude of scaled target amount. We observe very good agreement between predictions and experimental data for the spatiotemporal behavior of the coupled ITP and affinity process, and for key figures of merit, including scaled capture length and maximum capture efficiency. Lastly, we demonstrate that the resolution of ITP-AC increases linearly with time and purify 25 nt target DNA from 10 000-fold higher abundance background (contaminating) genomic fish sperm DNA. We perform this capture from 200 μL of sample in under 1 mm column length and within <10 min. PMID:24937777

  2. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  3. Column Liquid Chromatography.

    ERIC Educational Resources Information Center

    Majors, Ronald E.; And Others

    1984-01-01

    Reviews literature covering developments of column liquid chromatography during 1982-83. Areas considered include: books and reviews; general theory; columns; instrumentation; detectors; automation and data handling; multidimensional chromatographic and column switching techniques; liquid-solid chromatography; normal bonded-phase, reversed-phase,…

  4. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  5. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  6. Online micro-solid-phase extraction based on boronate affinity monolithic column coupled with high-performance liquid chromatography for the determination of monoamine neurotransmitters in human urine.

    PubMed

    Yang, Xiaoting; Hu, Yufei; Li, Gongke

    2014-05-16

    Quantification of monoamine neurotransmitters is very important in diagnosing and monitoring of patients with neurological disorders. We developed an online analytical method to selectively determine urinary monoamine neurotransmitters, which coupled the boronate affinity monolithic column micro-solid-phase extraction with high-performance liquid chromatography (HPLC). The boronate affinity monolithic column was prepared by in situ polymerization of vinylphenylboronic acid (VPBA) and N,N'-methylenebisacrylamide (MBAA) in a stainless capillary column. The prepared monolithic column showed good permeability, high extraction selectivity and capacity. The column-to-column reproducibility was satisfactory and the enrichment factors were 17-243 for four monoamine neurotransmitters. Parameters that influence the online extraction efficiency, including pH of sample solution, flow rate of extraction and desorption, extraction volume and desorption volume were investigated. Under the optimized conditions, the developed method exhibited low limit of detection (0.06-0.80μg/L), good linearity (with R(2) between 0.9979 and 0.9993). The recoveries in urine samples were 81.0-105.5% for four monoamine neurotransmitters with intra- and inter-day RSDs of 2.1-8.2% and 3.7-10.6%, respectively. The online analytical method was sensitive, accurate, selective, reliable and applicable to analysis of trace monoamine neurotransmitters in human urine sample.

  7. Rapid Screening of Drug-Protein Binding Using High-Performance Affinity Chromatography with Columns Containing Immobilized Human Serum Albumin

    PubMed Central

    Li, Ying-Fei; Zhang, Xiao-Qiong; Hu, Wei-Yu; Li, Zheng; Liu, Ping-Xia; Zhang, Zhen-Qing

    2013-01-01

    For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 − 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation. PMID:23607050

  8. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  9. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent. PMID:27289464

  10. Improving affinity chromatography resin efficiency using semi-continuous chromatography.

    PubMed

    Mahajan, Ekta; George, Anupa; Wolk, Bradley

    2012-03-01

    Protein A affinity chromatography is widely used for purification of monoclonal antibodies (MAbs) from harvested cell culture fluid (HCCF). At the manufacturing scale, the HCCF is typically loaded on a single Protein A affinity chromatography column in cycles until all of the HCCF is processed. Protein A resin costs are significant, comprising a substantial portion of the raw material costs in MAb manufacturing. Cost can be reduced by operating the process continuously using multiple smaller columns to a higher binding capacity in lieu of one industrial scale column. In this study, a series of experiments were performed using three 1-ml Hi-Trap™ MabSelect SuRe™ columns on a modified ÄKTA™ system operated according to the three Column Periodic Counter Current Chromatography (3C PCC) principle. The columns were loaded individually at different times until the 70% breakthrough point was achieved. The HCCF with unbound protein from the column was then loaded onto the next column to capture the MAb, preventing any protein loss. At any given point, all three columns were in operation, either loading or washing, enabling a reduction in processing time. The product yield and quality were evaluated and compared with a batch process to determine the effect of using the three column continuous process. The continuous operation shows the potential to reduce both resin volume and buffer consumption by ∼40%, however the system hardware and the process is more complex than the batch process. Alternative methods using a single standard affinity column, such as recycling load effluent back to the tank or increasing residence time, were also evaluated to improve Protein A resin efficiency. These alternative methods showed similar cost benefits but required longer processing time. PMID:22265178

  11. An Undergraduate Column Chromatography Experiment.

    ERIC Educational Resources Information Center

    Danot, M.; And Others

    1984-01-01

    Background information, list of materials needed, and procedures used are provided for an experiment designed to introduce undergraduate students to the theoretical and technical aspects of column chromatography. The experiment can also be shortened to serve as a demonstration of the column chromatography technique. (JN)

  12. "Clickable" agarose for affinity chromatography.

    PubMed

    Punna, Sreenivas; Kaltgrad, Eiton; Finn, M G

    2005-01-01

    Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

  13. Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts.

    PubMed

    Ciesla, L; Okine, M; Rosenberg, A; Dossou, K S S; Toll, L; Wainer, I W; Moaddel, R

    2016-01-29

    The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs. PMID:26774122

  14. The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells

    PubMed Central

    Bhatia, Prateek A.; Moaddel, Ruin; Wainer, Irving W.

    2010-01-01

    CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodopetra frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp c-DNA, using a baculovirus expression system. The resulting CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [3H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9MRP1) column, etoposide and furosemide on the CMAC(Sf9MRP2) column and etoposide and fumitremorgin C on the CMAC(Sf9BCPR) column The binding affinities (Ki values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [3H]-etoposide on the CMAC(Sf9MRP1) column to a greater extent than (R)-verapamil and the relative IC50 values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC50 values were consistent with previously reported data. The results indicated that the CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system. PMID:20441926

  15. The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells.

    PubMed

    Habicht, K-L; Frazier, C; Singh, N; Shimmo, R; Wainer, I W; Moaddel, R

    2013-01-01

    BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC-MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54 μM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized.

  16. Self-regenerating column chromatography

    DOEpatents

    Park, Woo K.

    1995-05-30

    The present invention provides a process for treating both cations and anions by using a self-regenerating, multi-ionic exchange resin column system which requires no separate regeneration steps. The process involves alternating ion-exchange chromatography for cations and anions in a multi-ionic exchange column packed with a mixture of cation and anion exchange resins. The multi-ionic mixed-charge resin column works as a multi-function column, capable of independently processing either cationic or anionic exchange, or simultaneously processing both cationic and anionic exchanges. The major advantage offered by the alternating multi-function ion exchange process is the self-regeneration of the resins.

  17. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  18. Affinity chromatography with an immobilized RNA enzyme.

    PubMed Central

    Vioque, A; Altman, S

    1986-01-01

    M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid. Images PMID:3526344

  19. 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

  20. Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with MS detection

    PubMed Central

    Temporini, C.; Pochetti, G.; Fracchiolla, G.; Piemontese, L.; Montanari, R.; Moaddel, R.; Laghezza, A.; Altieri, F.; Cervoni, L.; Ubiali, D.; Prada, E.; Loiodice, F.; Massolini, G.; Calleri, E.

    2013-01-01

    The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening towards PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of Frontal Affinity Chromatography coupled to Mass Spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments towards new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes. PMID:23466198

  1. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.

  2. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes. PMID:25764651

  3. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  4. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  5. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

  6. KINETIC STUDIES OF DRUG-PROTEIN INTERACTIONS BY USING PEAK PROFILING AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: EXAMINATION OF MULTI-SITE INTERACTIONS OF DRUGS WITH HUMAN SERUM ALBUMIN COLUMNS

    PubMed Central

    Tong, Zenghan; Schiel, John E.; Papastavros, Efthimia; Ohnmacht, Corey M.; Smith, Quentin R.; Hage, David S.

    2010-01-01

    Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (± 0.2) s-1 and 0.67 (± 0.04) s-1 at pH 7.4 and 37 °C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins. PMID:21067755

  7. Temperature programmable microfabricated gas chromatography column

    DOEpatents

    Manginell, Ronald P.; Frye-Mason, Gregory C.

    2003-12-23

    A temperature programmable microfabricated gas chromatography column enables more efficient chemical separation of chemical analytes in a gas mixture by the integration of a resistive heating element and temperature sensing on the microfabricated column. Additionally, means are provided to thermally isolate the heated column from their surroundings. The small heat capacity and thermal isolation of the microfabricated column improves the thermal time response and power consumption, both important factors for portable microanalytical systems.

  8. Manual Microscale Column Chromatography Pressurization Apparatus

    NASA Astrophysics Data System (ADS)

    Baldwin, Bruce W.

    2003-10-01

    Pressurization of a Pasteur pipet for microscale chromatography is simplified by connecting a 20- or 30-mL syringe to the pipet using a length of Tygon tubing. This simple system allows the student to easily dry-pack a column using common chromatography packing materials. Results were uniformly good for introductory, organic, or upper-division research chemistry students.

  9. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  10. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    PubMed

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  11. "Dry-column" chromatography of plant pigments

    NASA Technical Reports Server (NTRS)

    Woeller, F. H.; Lehwalt, M. F.; Oyama, V. I.

    1973-01-01

    Separation of plant pigments which can be accomplished on thin-layer silica plates with mixture of petroleum ether, halocarbon, acetone, and polar solvent can be readily translated into dry-column technique that yields reproducible chromatograms after elution in fashion of liquid chromatography with fluorimeter as detector. Best solvent system was found to be mixture of petroleum ether, dichloromethane, acetone, and ethyl acetate.

  12. Non-planar microfabricated gas chromatography column

    DOEpatents

    Lewis, Patrick R.; Wheeler, David R.

    2007-09-25

    A non-planar microfabricated gas chromatography column comprises a planar substrate having a plurality of through holes, a top lid and a bottom lid bonded to opposite surfaces of the planar substrate, and inlet and outlet ports for injection of a sample gas and elution of separated analytes. A plurality of such planar substrates can be aligned and stacked to provide a longer column length having a small footprint. Furthermore, two or more separate channels can enable multi-channel or multi-dimensional gas chromatography. The through holes preferably have a circular cross section and can be coated with a stationary phase material or packed with a porous packing material. Importantly, uniform stationary phase coatings can be obtained and band broadening can be minimized with the circular channels. A heating or cooling element can be disposed on at least one of the lids to enable temperature programming of the column.

  13. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  14. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  15. Site-specific DNA-affinity chromatography of the lac repressor.

    PubMed Central

    Herrick, G

    1980-01-01

    To test the feasibility of site-specific DNA-affinity chromatography, E. coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column. Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity. PMID:7001362

  16. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  17. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals. PMID:26952369

  18. Purification of glycolytic enzymes by using affinity-elution chromatography.

    PubMed Central

    Scopes, R K

    1977-01-01

    1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures. PMID:192194

  19. Affinity chromatography for purification of two urokinases from human urine.

    PubMed

    Takahashi, R; Akiba, K; Koike, M; Noguchi, T; Ezure, Y

    2000-05-26

    A new affinity chromatography (hydrophobic-mediated affinity chromatography), which was characterized by the matrix having both affinity site to urokinase and hydrophobic site, was established for the purification of urokinase from human urine. The hydrophobic affinity matrix (tentatively named PAS in the text) was prepared by immobilizing 6-aminocaproic acid on Sepharose CL-6B, followed by a coupling p-aminobenzamidine to a part of the hydrophobic site on the matrix. The PAS matrix was applied to the purification of urokinase from human urine, and high- and low-molecular weight pure urokinases were efficiently obtained in high yield by the present method. PMID:10892585

  20. Frontal affinity chromatography (FAC): theory and basic aspects.

    PubMed

    Kasai, Ken-ichi

    2014-01-01

    Frontal affinity chromatography (FAC) is a versatile analytical tool for determining specific interactions between biomolecules and is particularly useful in the field of glycobiology. This article presents its basic aspects, merits, and theory. PMID:25117240

  1. Affinity monolith chromatography: A review of principles and recent analytical applications

    PubMed Central

    Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

    2012-01-01

    Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

  2. Stable high capacity, F-actin affinity column

    SciTech Connect

    Luna, E.J.; Wang, Y.L.; Voss, E.W. Jr.; Branton, D.; Taylor, D.L.

    1982-11-10

    A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds /sup 125/I-heavy meromyosin and /sup 125/I-tropomyosin. The associations between the F-actin-binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

  3. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  4. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  5. Aptamer stationary phase for protein capture in affinity capillary chromatography.

    PubMed

    Connor, Adam C; McGown, Linda B

    2006-04-14

    The thrombin-binding DNA aptamer was used with thrombin as a model system to investigate protein capture using aptamer stationary phases in affinity capillary chromatography. The aptamer was covalently attached to the inner surface of a bare fused-silica glass capillary to serve as the stationary phase. Proteins were loaded onto the capillary via an applied pressure. The capillary was then washed to remove unbound and non-specifically associated proteins. Finally, the bound protein was released and eluted using 20 mM Tris buffer containing 8 M urea, pH 7.3, at 50 degrees C. Eluate was collected after each step (load, wash and elute) and relative amounts of protein each were compared using fluorescence spectroscopy. The identity of the protein in the collections was confirmed using matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The experiment was repeated for thrombin on a bare (unmodified) capillary and a capillary coated with a scrambled-sequence, non-G-quartet forming oligonucleotide that does not bind with thrombin. The results show that the aptamer stationary phase captures approximately three times as much thrombin as the control columns. The experiment was also repeated using human serum albumin (HSA) alone and in an equimolar mixture with thrombin. HSA was not retained on the aptamer capillary, nor did it affect the capture of thrombin from the mixture.

  6. Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

    PubMed

    Ahirwar, Rajesh; Nahar, Pradip

    2015-08-01

    Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A. PMID:26102634

  7. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  8. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column. PMID:19469504

  9. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency. PMID:25749943

  10. Evaluation and optimization of the metal-binding properties of a complex ligand for immobilized metal affinity chromatography.

    PubMed

    Chen, Bin; Li, Rong; Li, Shiyu; Chen, Xiaoli; Yang, Kaidi; Chen, Guoliang; Ma, Xiaoxun

    2016-02-01

    The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors. PMID:26632098

  11. Specific capture of uranyl protein targets by metal affinity chromatography.

    PubMed

    Basset, Christian; Dedieu, Alain; Guérin, Philippe; Quéméneur, Eric; Meyer, Daniel; Vidaud, Claude

    2008-03-28

    To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO2(2+)) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis. PMID:18308325

  12. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    PubMed

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-01

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods. PMID:26973166

  13. Ion Exchange and Liquid Column Chromatography.

    ERIC Educational Resources Information Center

    Walton, Harold F.

    1980-01-01

    Emphasizes recent advances in principles and methodology in ion exchange and chromatography. Two tables list representative examples for inorganic ions and organic compounds. Cites 544 references. (CS)

  14. A Better Method for Filling Pasteur Pipet Chromatography Columns

    ERIC Educational Resources Information Center

    Ruekberg, Ben

    2006-01-01

    An alternative method for the preparation of Pasteur pipet chromatography columns is presented that allows the column to be filled with solvent without bubbles and allows greater control of fluid flow while the materials to be separated are added. Students are required to wear gloves and goggles and caution should be used while handling glass…

  15. Combining micro dry column chromatography and mass spectrometry

    NASA Technical Reports Server (NTRS)

    Bauman, A. J.

    1970-01-01

    Dry column chromatography principles applied in microscale produce technique to minimize time in preparing and analyzing colorless constituents of soluble mixtures. Glass pipette microcolumns filled with finely sieved adsorbents permit capillary attraction and separation in 3 to 15 minutes. Technique is adaptable to gas chromatography.

  16. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  17. Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Edwards, Katarina; Eriksson, Jonny; Ohlson, Sten; Ying, Janet To Yiu; Torres, Jaume; Hernández, Víctor Agmo

    2016-02-01

    Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures. PMID:26673836

  18. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  19. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  20. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.

  1. Design of affinity tags for one-step protein purification from immobilized zinc columns

    SciTech Connect

    Pasquinelli, R.S.; Shepherd, R.E.; Koepsel, R.R.; Zhao, A.; Ataai, M.M.

    2000-02-01

    Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to e superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. for example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper the authors have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

  2. A fullerene C60-based ligand in a stationary phase for affine chromatography of membrane porphyrin-binding proteins

    NASA Astrophysics Data System (ADS)

    Amirshakhi, N.; Alyautdin, R. N.; Orlov, A. P.; Poloznikov, A. A.; Kuznetsov, D. A.

    2008-11-01

    A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used.

  3. High speed immuno-affinity chromatography on supports with gigapores and porous glass.

    PubMed

    Schuste, M; Wasserbauer, E; Neubauer, A; Jungbauer, A

    2000-01-01

    Immuno-affinity chromatography exploiting the Ca2+ dependent interaction of the anti-Flag antibody and Flag-tagged proteins has been investigated. The antibody has been immobilized on porous glass beads (Prosep) containing gigapores and on a monolith, the polymethacrylate based Convective Interactive Media (CIM) column at a ligand density of 2 mg/g and 10 mg/ml respectively. The performance of the columns was assessed by applying clarified yeast culture supernatant containing overexpressed Flag-human serum albumin. Dynamic binding capacity and purity was checked at various flow rates ranging from 100 cm/h to 800 cm/h. 95% purity could be obtained. Anti Flag-CIM columns showed a higher unspecific adsorption, requiring a longer wash cycle to obtain the same purity compared to the Prosep column. Anti Flag-CIM columns showed a flow independent performance, which is explained by its monolithic structure. A decreasing dynamic binding capacity with flow was observed with anti-Flag-Prosep columns. Both columns are suited to purify milligrams of protein out of a yeast culture supernatant within a few minutes. We considered them as promising candidates for high throughput screening, where fast purification is a necessity.

  4. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  5. Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

    PubMed Central

    Clarke, C M; Knowles, J R

    1977-01-01

    The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed. Images Fig. 4. Fig. 7. PMID:597251

  6. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high-performance affinity chromatography.

    PubMed

    Zhang, Jiwen; Li, Haiyan; Sun, Lixin; Wang, Caifen

    2015-01-01

    The kinetics of the association and dissociation are fundamental kinetic processes for the host-guest interactions (such as the drug-target and drug-excipient interactions) and the in vivo performance of supramolecules. With advantages of rapid speed, high precision and ease of automation, the high-performance affinity chromatography (HPAC) is one of the best techniques to measure the interaction kinetics of weak to moderate affinities, such as the typical host-guest interactions of drug and cyclodextrins by using a cyclodextrin-immobilized column. The measurement involves the equilibration of the cyclodextrin column, the upload and elution of the samples (non-retained substances and retained solutes) at different flow rates on the cyclodextrin and control column, and data analysis. It has been indicated that cyclodextrin-immobilized chromatography is a cost-efficient high-throughput tool for the measurement of (small molecule) drug-cyclodextrin interactions as well as the dissociation of other supramolecules with relatively weak, fast, and extensive interactions. PMID:25749964

  7. [Progresses in screening active compounds from herbal medicine by affinity chromatography].

    PubMed

    Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

    2015-03-01

    Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening. PMID:26226740

  8. Separation by column chromatography of cells active in delayed-onset hypersensitivities.

    PubMed Central

    Godfrey, H P; Gell, P G

    1976-01-01

    Lymph node cells from guinea-pigs contact sensitive to 1-thiocyamo-2,4-dinitrobenzene have been fractionated by affinity chromatography over modified polyacrylamide beads. Cells mediating lymphokine release in response to active sensitizer were depleted only by chromatography over dinitrophyenyl (DNP) containing substrates and could be specifically eluted with DNP-glycine. DNP rosette-forming cells (RFC) were equally well depleted by chromatography using either DNP or trinitrophenyl containing materials but could not be eluted from the columns by DNP-glycine. While the antigen receptors of cells mediating the release of macrophage agglutination factor in response to DNP-containing antigens and of DNP-RFC were found to be hapten-specific, their specificity was shown to differ using chromatography over trinitrophenyl containing polyacrylamide. PMID:776818

  9. Procedure for rapid isolation of photosynthetic reaction centers using cytochrome c affinity chromatography

    SciTech Connect

    Brudvig, G.W.; Worland, S.T.; Sauer, K.

    1983-02-01

    Horse heart cytochrome c linked to Sepharose 4B is used to purify reaction centers from Rhodopseudomonas sphaeroides R-26. This procedure allows for an initial recovery of 80-90% of the bacterial reaction centers present in chromatophore membranes. High purity reaction centers (A/sub 280//A/sub 802/ < 1.30) can be obtained with a 30% recovery. Reaction centers from wild-type Rps. sphaeroides and Rps. capsulata also bind to a cytochrome c column. Cytochrome c affinity chromatography can also be used to isolate photosystem I complexes from spinach chloroplasts.

  10. Column precipitation chromatography: an approach to quantitative analysis of eigencolloids.

    PubMed

    Breynaert, E; Maes, A

    2005-08-01

    A new column precipitation chromatography (CPC) technique, capable of quantitatively measuring technetium eigencolloids in aqueous solutions, is presented. The CPC technique is based on the destabilization and precipitation of eigencolloids by polycations in a confined matrix. Tc(IV) colloids can be quantitatively determined from their precipitation onto the CPC column (separation step) and their subsequent elution upon oxidation to pertechnetate by peroxide (elution step). A clean-bed particle removal model was used to explain the experimental results. PMID:16053321

  11. Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.

    PubMed

    Kennedy, Jacob J; Yan, Ping; Zhao, Lei; Ivey, Richard G; Voytovich, Uliana J; Moore, Heather D; Lin, Chenwei; Pogosova-Agadjanyan, Era L; Stirewalt, Derek L; Reding, Kerryn W; Whiteaker, Jeffrey R; Paulovich, Amanda G

    2016-02-01

    A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847

  12. 3D printed metal columns for capillary liquid chromatography.

    PubMed

    Sandron, S; Heery, B; Gupta, V; Collins, D A; Nesterenko, E P; Nesterenko, P N; Talebi, M; Beirne, S; Thompson, F; Wallace, G G; Brabazon, D; Regan, F; Paull, B

    2014-12-21

    Coiled planar capillary chromatography columns (0.9 mm I.D. × 60 cm L) were 3D printed in stainless steel (316L), and titanium (Ti-6Al-4V) alloys (external dimensions of ~5 × 30 × 58 mm), and either slurry packed with various sized reversed-phase octadecylsilica particles, or filled with an in situ prepared methacrylate based monolith. Coiled printed columns were coupled directly with 30 × 30 mm Peltier thermoelectric direct contact heater/cooler modules. Preliminary results show the potential of using such 3D printed columns in future portable chromatographic devices. PMID:25285334

  13. Effect of column dimension on observed column efficiency in very high pressure liquid chromatography.

    PubMed

    Wu, Naijun; Bradley, Ashley C

    2012-10-26

    The effect of extra-column volume on observed linear velocity was investigated for columns of various internal diameters in very high pressure liquid chromatography. The results showed that the observed linear velocities were approximately 4.5, 9.5, 16.8, and 39.5% lower than the linear velocities corrected for the extra-column volume contribution for 4.6, 3.0, 2.1, and 1.0mm internal diameter columns, respectively. An empirical relationship between extra-column band broadening and extra-column volume was obtained using 50 cm long tubings of various internal diameters. The peak variance from the extra-column volume is near linearly proportional to the square of the extra-column volume for tubings with 0.0635-0.178 mm (0.025-0.07 in.) i.d. using a 50/50 acetonitrile/water mobile phase at flow rates greater than 0.3 mL/min. The effect of column internal diameter and column length on observed efficiency was studied using 50mm columns with four different column internal diameters and 2.1mm i.d columns with three different lengths. The results showed that the observed column efficiencies for 3.0, 2.1, and 1.0mm internal diameter columns were 18, 33, and 73% lower than that for a 4.6mm internal diameter column for benzophenone (k=5.5), respectively. An approximate 20% decrease in theoretical plate number was observed for propiophenone (k=3.3) using a 50 mm × 2.1 mm column packed with 1.7 μm particles compared to a 150 mm × 2.1 mm column packed with 5.0 μm particles, while the former column provided 9 fold faster separation. It is the column to extra column volume ratio instead of absolute extra-column volume that determines the degree of extra-column band-broadening in VHPLC.

  14. Molecular modeling of the affinity chromatography of monoclonal antibodies.

    PubMed

    Paloni, Matteo; Cavallotti, Carlo

    2015-01-01

    Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein-ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A procedure to extend the proposed methodology to include the effect of the environment (buffer solution, spacer, support matrix) is also briefly outlined. PMID:25749965

  15. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

    PubMed Central

    Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

    2013-01-01

    The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

  16. Column Chromatography To Obtain Organic Cation Sorption Isotherms.

    PubMed

    Jolin, William C; Sullivan, James; Vasudevan, Dharni; MacKay, Allison A

    2016-08-01

    Column chromatography was evaluated as a method to obtain organic cation sorption isotherms for environmental solids while using the peak skewness to identify the linear range of the sorption isotherm. Custom packed HPLC columns and standard batch sorption techniques were used to intercompare sorption isotherms and solid-water sorption coefficients (Kd) for four organic cations (benzylamine, 2,4-dichlorobenzylamine, phenyltrimethylammonium, oxytetracycline) with two aluminosilicate clay minerals and one soil. A comparison of Freundlich isotherm parameters revealed isotherm linearity or nonlinearity was not significantly different between column chromatography and traditional batch experiments. Importantly, skewness (a metric of eluting peak symmetry) analysis of eluting peaks can establish isotherm linearity, thereby enabling a less labor intensive means to generate the extensive data sets of linear Kd values required for the development of predictive sorption models. Our findings clearly show that column chromatography can reproduce sorption measures from conventional batch experiments with the benefit of lower labor-intensity, faster analysis times, and allow for consistent sorption measures across laboratories with distinct chromatography instrumentation. PMID:27379799

  17. Column Chromatography To Obtain Organic Cation Sorption Isotherms.

    PubMed

    Jolin, William C; Sullivan, James; Vasudevan, Dharni; MacKay, Allison A

    2016-08-01

    Column chromatography was evaluated as a method to obtain organic cation sorption isotherms for environmental solids while using the peak skewness to identify the linear range of the sorption isotherm. Custom packed HPLC columns and standard batch sorption techniques were used to intercompare sorption isotherms and solid-water sorption coefficients (Kd) for four organic cations (benzylamine, 2,4-dichlorobenzylamine, phenyltrimethylammonium, oxytetracycline) with two aluminosilicate clay minerals and one soil. A comparison of Freundlich isotherm parameters revealed isotherm linearity or nonlinearity was not significantly different between column chromatography and traditional batch experiments. Importantly, skewness (a metric of eluting peak symmetry) analysis of eluting peaks can establish isotherm linearity, thereby enabling a less labor intensive means to generate the extensive data sets of linear Kd values required for the development of predictive sorption models. Our findings clearly show that column chromatography can reproduce sorption measures from conventional batch experiments with the benefit of lower labor-intensity, faster analysis times, and allow for consistent sorption measures across laboratories with distinct chromatography instrumentation.

  18. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  19. Kinetic analysis of drug-protein interactions by affinity chromatography.

    PubMed

    Bi, Cong; Beeram, Sandya; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-10-01

    Information on the kinetics of drug-protein interactions is of crucial importance in drug discovery and development. Several methods based on affinity chromatography have been developed in recent years to examine the association and dissociation rates of these processes. These techniques include band-broadening measurements, the peak decay method, peak fitting methods, the split-peak method, and free fraction analysis. This review will examine the general principles and applications of these approaches and discuss their use in the characterization, screening and analysis of drug-protein interactions in the body. PMID:26724332

  20. Enrichment of Phosphopeptides via Immobilized Metal Affinity Chromatography.

    PubMed

    Swaney, Danielle L; Villén, Judit

    2016-03-01

    Immobilized metal affinity chromatography (IMAC) is a frequently used method for the enrichment of phosphorylated peptides from complex, cellular lysate-derived peptide mixtures. Here we outline an IMAC protocol that uses iron-chelated magnetic beads to selectively isolate phosphorylated peptides for mass spectrometry-based proteomic analysis. Under acidic conditions, negatively charged phosphoryl modifications preferentially bind to positively charged metal ions (e.g., Fe(3+), Ga(3+)) on the beads. After washing away nonphosphorylated peptides, a pH shift to basic conditions causes the elution of bound phosphopeptides from the metal ion. Under optimal conditions, very high specificity for phosphopeptides can be achieved. PMID:26933247

  1. Affinity chromatography of human leukocyte and diploid cell interferons on sepharose-bound antibodies.

    PubMed

    Berg, K; Ogburn, C A; Paucker, K; Mogensen, K E; Cantell, K

    1975-02-01

    Interferons produced in human peripheral leukocytes (LE) and foreskin fibroblast (FS-4) cells were subjected to affinity chromatography on Sepharose-bound globulins from rabbits immunized with these interferons. Anti-LE interferon sera neutralized both interferons, but titers against FS-4 interferon were consistently lower than those against LE interferon. Anti-FS-4 interferon sera neutralized only FS-4 but not LE interferon. Accordingly, affinity columns constructed with anti-FS-4 globulin excluded LE but not FS-4 interferon, whereas those prepared with anti-LE interferon globulin bound and eluted both LE and FS-4 interferons. Purification of native interferons of both types on anti-LE interferon-Sepharose ranged from 680- to 3,600-fold and recoveries from 72 to 126%. Specific activities of eluate pools varied from 4 to 30 times 10-6 reference (B, 69/19) units per milligram protien.

  2. Evaluating two process scale chromatography column header designs using CFD.

    PubMed

    Johnson, Chris; Natarajan, Venkatesh; Antoniou, Chris

    2014-01-01

    Chromatography is an indispensable unit operation in the downstream processing of biomolecules. Scaling of chromatographic operations typically involves a significant increase in the column diameter. At this scale, the flow distribution within a packed bed could be severely affected by the distributor design in process scale columns. Different vendors offer process scale columns with varying design features. The effect of these design features on the flow distribution in packed beds and the resultant effect on column efficiency and cleanability needs to be properly understood in order to prevent unpleasant surprises on scale-up. Computational Fluid Dynamics (CFD) provides a cost-effective means to explore the effect of various distributor designs on process scale performance. In this work, we present a CFD tool that was developed and validated against experimental dye traces and tracer injections. Subsequently, the tool was employed to compare and contrast two commercially available header designs.

  3. Evaluating two process scale chromatography column header designs using CFD.

    PubMed

    Johnson, Chris; Natarajan, Venkatesh; Antoniou, Chris

    2014-01-01

    Chromatography is an indispensable unit operation in the downstream processing of biomolecules. Scaling of chromatographic operations typically involves a significant increase in the column diameter. At this scale, the flow distribution within a packed bed could be severely affected by the distributor design in process scale columns. Different vendors offer process scale columns with varying design features. The effect of these design features on the flow distribution in packed beds and the resultant effect on column efficiency and cleanability needs to be properly understood in order to prevent unpleasant surprises on scale-up. Computational Fluid Dynamics (CFD) provides a cost-effective means to explore the effect of various distributor designs on process scale performance. In this work, we present a CFD tool that was developed and validated against experimental dye traces and tracer injections. Subsequently, the tool was employed to compare and contrast two commercially available header designs. PMID:24616438

  4. 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

  5. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  6. Comparison of Inlet Geometry in Microfluidic Cell Affinity Chromatography

    PubMed Central

    Li, Peng; Tian, Yu; Pappas, Dimitri

    2011-01-01

    Cell separation based on microfluidic affinity chromatography is a widely used methodology in cell analysis research when rapid separations with high purity are needed. Several successful examples have been reported with high separation efficiency and purity; however, cell capture at the inlet area and inlet design has not been extensively described or studied. The most common inlets—used to connect the microfluidic chip to pumps, tubing, etc—are vertical (top-loading) inlets and parallel (in-line) inlets. In this work, we investigated the cell capture behavior near the affinity chip inlet area and compared the different performance of vertical inlet devices and parallel inlet devices. Vertical inlet devices showed significant cell capture capability near the inlet area, which led to the formation of cell blockages as the separation progressed. Cell density near the inlet area was much higher than the remaining channel, while for parallel inlet chips cell density at the inlet area was similar to the rest of the channel. In this paper, we discuss the effects of inlet type on chip fabrication, nonspecific binding, cell capture efficiency, and separation purity. We also discuss the possibility of using vertical inlets in negative selection separations. Our findings show that inlet design is critical and must be considered when fabricating cell affinity microfluidic devices. PMID:21207967

  7. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Graça, Vânia C; Sousa, Fani; Santos, Paulo F; Almeida, Paulo S

    2015-01-01

    Affinity chromatography (AC) is one of the most important techniques for the separation and purification of biomolecules, being probably the most selective technique for protein purification. It is based on unique specific reversible interactions between the target molecule and a ligand. In this affinity interaction, the choice of the ligand is extremely important for the success of the purification protocol. The growing interest in AC has motivated an intense research effort toward the development of materials able to overcome the disadvantages of conventional natural ligands, namely their high cost and chemical and biological lability. In this context, synthetic dyes have emerged, in recent decades, as a promising alternative to biological ligands. Herein, detailed protocols for the assembling of a new chromatographic dye-ligand affinity support bearing an immobilized aminosquarylium cyanine dye on an agarose-based matrix (Sepharose CL-6B) and for the separation of a mixture o f three standard proteins: lysozyme, α-chymotrypsin, and trypsin are provided. PMID:25749942

  8. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

    André, C; De Backer, J P; Guillet, J C; Vanderheyden, P; Vauquelin, G; Strosberg, A D

    1983-01-01

    Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein. Images Fig. 3. PMID:6605245

  9. Packed column supercritical fluid chromatography using deactivated stationary phases

    SciTech Connect

    Ashraf-Khorassani, M.; Taylor, L.T.; Henry, R.A.

    1988-08-01

    A new cross-linked cyanopropyl bonded phase silica (Delta-bond) has been studied as a stationary phase for packed column supercritical fluid chromatography of basic nitrogen-containing compounds. The bonded phase impedes access to uncapped silanol sites, thereby giving rise to better peak shapes and more rapid elution without the necessity of a polar modifier in the mobile phase. Experiments both at elevated temperature and in the presence of a methanol modifier revealed that there is no short- or long-term deleterious effect on the column as opposed to the conventional cyanopropyl phase.

  10. Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

    PubMed

    Ulbrich, N; Lin, A; Wool, I G

    1979-09-10

    The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column. PMID:468846

  11. Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns.

    PubMed

    Jones, Andrew; Pravadali-Cekic, Sercan; Hua, Stanley; Kocic, Danijela; Camenzuli, Michelle; Dennis, Gary; Shalliker, Andrew

    2016-04-26

    A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

  12. Search for Amyloid-Binding Proteins by Affinity Chromatography

    PubMed Central

    Calero, Miguel; Rostagno, Agueda; Ghiso, Jorge

    2013-01-01

    ‘Amyloid binging proteins’ is a generic term used to designate proteins that interact with different forms of amyloidogenic peptides or proteins and that, as a result, may modulate their physiological and pathological functions by altering solubility, transport, clearance, degradation, and fibril formation. We describe a simple affinity chromatography protocol to isolate and characterize amyloid-binding proteins based on the use of sequential elution steps that may provide further information on the type of binding interaction. As an example, we depict the application of this protocol to the study of Alzheimer’s amyloid β (Aβ) peptide-binding proteins derived from human plasma. Biochemical analysis of the proteins eluted under different conditions identified serum amyloid P component (SAP) and apolipoprotein J (clusterin) as the main plasma Aβ-binding proteins while various apolipoproteins (apoA-IV, apoE, and apoA-I), as well as albumin (HSA) and fibulin were identified as minor contributors. PMID:22528093

  13. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119

  14. Production and Purification of Streptokinase by Protected Affinity Chromatography

    PubMed Central

    Babashamsi, Mohammad; Razavian, Mohammad Hossein; Nejadmoghaddam, Mohammad Reza

    2009-01-01

    Streptokinase is an extracellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy. The rate of bacterial growth and streptokinase production was studied in condition of excess glucose addition to culture media and its pH maintenance. The streptokinase product of the bacterial culture was preliminary extracted by salt precipitation and then purified by affinity chromatography on plasminogen substituted sepharose-4B in a condition that the plasminogen active site was protected from streptokinase-induced activation. The purity of streptokinase was confirmed by SDS-PAGE and its biological activity determined in a specific streptokinase assay. The results showed that in the fed–batch culture, the rate of streptokinase production increased over two times as compared with the batch culture while at the same time, shortening the streptokinase purification to a single step increased the yield over 95% at the chromatography stage. PMID:23407807

  15. Affinity chromatography of branched oligosaccharides in rat liver beta-glucuronidase.

    PubMed

    Hoja-Lukowicz, D; Lityńska, A; Wójczyk, B S

    2001-05-01

    Rat liver microsomal and lysosomal beta-glucuronidase-derived glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. These glycopeptides were studied by sequential chromatography on lectin-affinity columns such as concanavalin A, lentil lectin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin I, Triticum vulgaris agglutinin, Glycine max agglutinin and Ulex europaeus agglutinin. Using serial lectin affinity chromatography approach combined with neuraminidase treatment allowed us to show the unexpected presence of complex tri- and/or tetraantennary type glycans (40.8 and 17.0% for microsomal and lysosomal enzyme, respectively). Moreover, the application of neuraminidase treatment revealed that complex biantennary type glycans, present on lysosomal beta-glucuronidase, are almost fully sialylated while the same type of glycans present on microsomal enzyme do not contain sialic acid. Furthermore, the results obtained confirmed that microsomal and lysosomal beta-glucuronidases possess high mannose and/or hybrid type glycans (19.6 and 36.6%, respectively), and complex biantennary type glycans (38.9 and 46.4%, respectively). PMID:11393703

  16. Fiber-based monolithic columns for liquid chromatography.

    PubMed

    Ladisch, Michael; Zhang, Leyu

    2016-10-01

    Fiber-based monoliths for use in liquid chromatographic separations are defined by columns packed with aligned fibers, woven matrices, or contiguous fiber structures capable of achieving rapid separations of proteins, macromolecules, and low molecular weight components. A common denominator and motivating driver for this approach, first initiated 25 years ago, was reducing the cost of bioseparations in a manner that also reduced residence time of retained components while achieving a high ratio of mass to momentum transfer. This type of medium, when packed into a liquid chromatography column, minimized the fraction of stagnant liquid and resulted in a constant plate height for non-adsorbing species. The uncoupling of dispersion from eluent flow rate enabled the surface chemistry of the stationary phase to be considered separately from fluid transport phenomena and pointed to new ways to apply chemistry for the engineering of rapid bioseparations. This paper addresses developments and current research on fiber-based monoliths and explains how the various forms of this type of chromatographic stationary phase have potential to provide new tools for analytical and preparative scale separations. The different stationary phases are discussed, and a model that captures the observed constant plate height as a function of mobile phase velocity is reviewed. Methods that enable hydrodynamically stable fiber columns to be packed and operated over a range of mobile phase flow rates, together with the development of new fiber chemistries, are shown to provide columns that extend the versatility of liquid chromatography using monoliths, particularly at the preparative scale. Graphical Abstract Schematic representation of a sample mixture being separated by a rolled-stationary phase column, resulting separated peaks shown in the chromatogram. PMID:27553948

  17. Purification of infective bluetongue virus particles by immuno-affinity chromatography using anti-core antibody.

    PubMed

    Chand, Karam; Biswas, Sanchay K; Mondal, Bimalendu

    2016-03-01

    An immuno-affinity chromatography technique for purification of infective bluetongue virus (BTV) has been descried using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen-antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material was tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. This method was applied for selective capture of BTV from a mixture of other viruses. As group-specific antibodies (against BTV core) were used to capture the virus, it is expected that virus of all BTV serotypes could be purified by this method. This method will be helpful for selective capture and enrichment of BTV from concurrently infected blood or tissue samples for efficient isolation in cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation. PMID:26925450

  18. Automated hydrophobic interaction chromatography column selection for use in protein purification.

    PubMed

    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 ((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

  19. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

    PubMed

    DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U

    2016-05-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

  20. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  1. Direct probing of chromatography columns by laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    McGuffin, V. L.

    1992-12-01

    This report summarizes the progress and accomplishments of this research project from 1 Sep. 1989 to 28 Feb. 1993. During this period, we have accomplished all of the primary scientific objectives of the research proposal: (1) constructed and evaluated a laser-induced fluorescence detection system that allows direct examination of the chromatographic column, (2) examined nonequilibrium processes that occur upon solute injection and elution, (3) examined solute retention in liquid chromatography as a function of temperature and pressure, (4) examined solute zone dispersion in liquid chromatography as a function of temperature and pressure, and (5) developed appropriate theoretical models to describe these phenomena. In each of these studies, substantial knowledge has been gained of the fundamental processes that are responsible for chromatographic separations. In addition to these primary research objectives, we have made significant progress in three related areas: (1) examined pyrene as a fluorescent polarity probe in supercritical fluids and liquids as a function of temperature and pressure, (2) developed methods for the class-selective identification of polynuclear aromatic hydrocarbons in coal-derived fluids by microcolumn liquid chromatography with fluorescence quenching detection, and (3) developed methods for the determination of saturated and unsaturated (including omega-3) fatty acids in fish oil extracts by microcolumn liquid chromatography with laser-induced fluorescence detection. In these studies, the advanced separation and detection techniques developed in our laboratory are applied to practical problems of environmental and biomedical significance.

  2. Direct probing of chromatography columns by laser-induced fluorescence

    SciTech Connect

    McGuffin, V.L.

    1992-12-07

    This report summarizes the progress and accomplishments of this research project from September 1, 1989 to February 28, 1993. During this period, we have accomplished all of the primary scientific objectives of the research proposal: (1) constructed and evaluated a laser-induced fluorescence detection system that allows direct examination of the chromatographic column, (2) examined nonequilibrium processes that occur upon solute injection and elution, (3) examined solute retention in liquid chromatography as a function of temperature and pressure, (4) examined solute zone dispersion in liquid chromatography as a function of temperature and pressure, and (5) developed appropriate theoretical models to describe these phenomena. In each of these studies, substantial knowledge has been gained of the fundamental processes that are responsible for chromatographic separations. In addition to these primary research objectives, we have made significant progress in three related areas: (1) examined pyrene as a fluorescent polarity probe insupercritical fluids and liquids as a function of temperature and pressure, (2) developed methods for the class-selective identification of polynuclear aromatic hydrocarbons in coal-derived fluids by microcolumn liquid chromatography with fluorescence quenching detection, and (3) developed methods for the determination of saturated and unsaturated (including omega-3) fatty acids in fish oil extracts by microcolumn liquid chromatography with laser-induced fluorescence detection. In these studies, the advanced separation and detection techniques developed in our laboratory are applied to practical problems of environmental and biomedical significance.

  3. If You Were a Molecule in a Chromatography Column, What Would You See?

    ERIC Educational Resources Information Center

    Mattice, John

    2008-01-01

    To visualize what takes place in a chromatography column, enlarge the molecules to human size and expand the columns to keep the ratio of size of molecule to size of column the same. If we were molecules, what would the columns be like? A typical gas chromatography (GC) capillary column would be 50 x 10 [superscript 6] 6 km (31 million mi) long,…

  4. Mining the soluble chloroplast proteome by affinity chromatography

    PubMed Central

    Bayer, Roman G; Stael, Simon; Csaszar, Edina; Teige, Markus

    2011-01-01

    Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO2, they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions. PMID:21365755

  5. Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

    PubMed

    Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

    2012-11-01

    Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential. PMID:22918538

  6. Column properties and flow profiles of a flat, wide column for high-pressure liquid chromatography

    SciTech Connect

    Mriziq, Khaled S; Guiochon, Georges A

    2008-01-01

    The design and the construction of a pressurized, flat, wide column for high-performance liquid chromatography (HPLC) are described. This apparatus, which is derived from instruments that implement over-pressured thin layer chromatography, can carry out only uni-dimensional chromatographic separations. However, it is intended to be the first step in the development of more powerful instruments that will be able to carry out two-dimensional chromatographic separations, in which case, the first separation would be a space-based separation, LC{sup x}, taking place along one side of the bed and the second separation would be a time-based separation, LC{sup t}, as in classical HPLC but proceeding along the flat column, not along a tube. The apparatus described consists of a pressurization chamber made of a Plexiglas block and a column chamber made of stainless steel. These two chambers are separated by a thin Mylar membrane. The column chamber is a cavity which is filled with a thick layer (ca. 1 mm) of the stationary phase. Suitable solvent inlet and outlet ports are located on two opposite sides of the sorbent layer. The design allows the preparation of a homogenous sorbent layer suitable to be used as a chromatographic column, the achievement of effective seals of the stationary phase layer against the chamber edges, and the homogenous flow of the mobile phase along the chamber. The entire width of the sorbent layer area can be used to develop separations or elute samples. The reproducible performance of the apparatus is demonstrated by the chromatographic separations of different dyes. This instrument is essentially designed for testing detector arrays to be used in a two-dimensional LC{sup x} x LC{sup t} instrument. The further development of two-dimension separation chromatographs based on the apparatus described is sketched.

  7. SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications.

    PubMed

    Haney, Paul J; Draveling, Connie; Durski, Wendy; Romanowich, Kathryn; Qoronfleh, M Walid

    2003-04-01

    Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry. PMID:12699691

  8. Performance of the same column in supercritical fluid chromatography and in liquid chromatography.

    PubMed

    Lambert, Nándor; Felinger, Attila

    2015-08-28

    We have studied the chromatographic behavior of the homologous series of alkylbenzenes (ranging from octylbenzene to octadecylbenzene) on the same C18 reversed-phase column in supercritical fluid chromatography (SFC) and reversed phase liquid chromatography (RPLC) at various experimental conditions, such as different eluent compositions, flow-rates, and mobile phase densities. The first and the second moments of the peaks were used to estimate the overall mass-transfer processes in both chromatographic modes using the stochastic model of chromatography. The results confirm that in SFC - as the density of the mobile phase is influenced by the flow-rate - there is a broader variation of mass-transfer properties than in liquid chromatography. As expected, the optimum mobile phase velocity is higher in SFC, but there is no real difference in the minimum value of plate height, i.e. in the optimum efficiency.

  9. Group type analysis of asphalt by column liquid chromatography

    SciTech Connect

    Zhang, C.; Yang, J.; Xue, Y.; Li, Y.

    2008-07-01

    An improved analysis method for characterization of asphalt was established. The method is based on column chromatography technique. The asphalts were separated into four groups: saturates, aromatics, resins, and asphaltenes, quantitatively. About 0.1 g of sample was required in each analysis. About 20 mL of n-heptanes was used to separate out saturates first. Then about 35 mL of n-heptanes/dichloromethane (.5, v/v) mixture was used to separate out aromatics. About 30 mL of dichloromethane/tetrahydrofuran (1/3, v/v) mixture was used to separate out resin. The quality of the separation was confirmed by infrared spectra (IR) and {sup 1}H NMR analysis. The model compounds, tetracosan for saturates, dibenz(o)anthracen for aromatics, and acetanilide for resins were used for verification. The IR and {sup 1}H NMR analysis of the prepared fractions from the column liquid chromatography were in good agreement that of pure reagents.

  10. Methodology for optimally sized centrifugal partition chromatography columns.

    PubMed

    Chollet, Sébastien; Marchal, Luc; Jérémy Meucci; Renault, Jean-Hugues; Legrand, Jack; Foucault, Alain

    2015-04-01

    Centrifugal Partition Chromatography (CPC) is a separation process based on the partitioning of solutes between two partially miscible liquid phases. There is no solid support for the stationary phase. The centrifugal acceleration is responsible for both stationary phase retention and mobile phase dispersion. CPC is thus a process based on liquid-liquid mass transfer. The separation efficiency is mainly influenced by the hydrodynamics of the phases in each cell of the column. Thanks to a visualization system, called "Visual CPC", it was observed that the mobile phase can flow through the stationary phase as a sheet, or a spray. Hydrodynamics, which directly governs the instrument efficiency, is directly affected during scale changes, and non-linear phenomena prevent the successful achievement of mastered geometrical scale changes. In this work, a methodology for CPC column sizing is proposed, based on the characterization of the efficiency of advanced cell shapes, taking into account the hydrodynamics. Knowledge about relationship between stationary phase volume, cell efficiency and separation resolution in CPC allowed calculating the optimum cell number for laboratory and industrial scale CPC application. The methodology is highlighted with results on five different geometries from 25 to 5000 mL, for two applications: the separation of alkylbenzene by partitioning with heptane/methanol/water biphasic system; and the separation of peptides by partitioning with n-butanol/acetic acid/water (4/1/5) biphasic system. With this approach, it is possible to predict the optimal CPC column length leading to highest productivity.

  11. Affinity selection-based two-dimensional chromatography coupled with high-performance liquid chromatography-mass spectrometry for discovering xanthine oxidase inhibitors from Radix Salviae Miltiorrhizae.

    PubMed

    Fu, Yu; Mo, Hua-Yan; Gao, Wen; Hong, Jia-Ying; Lu, Jun; Li, Ping; Chen, Jun

    2014-08-01

    Xanthine oxidase (XOD) is a key oxidative enzyme to the pathogenesis of hyperuricemia and certain diseases induced by excessive reactive oxygen species. XOD inhibitors could provide an important therapeutic approach to treat such diseases. A new method using affinity selection-based two-dimensional chromatography coupled with liquid chromatography-mass spectrometry was developed for the online screening of potential XOD inhibitors from Radix Salviae Miltiorrhizae. Based on our previous study, the two-dimensional, turbulent-flow chromatography (TFC) was changed to a mixed-mode anion-exchange/reversed-phase column and one reversed-phase column. The developed method was validated to be selective and sensitive for screening XOD-binding compounds, especially weak acidic ones, in the extracts. Three salvianolic acids were screened from the Radix Salviae Miltiorrhizae extract via the developed method. The XOD inhibitory activities of salvianolic acid C and salvianolic acid A were confirmed, and their inhibitory modes were measured. Salvianolic acid C exhibited potent XOD inhibitory activity with an IC(50) of 9.07 μM. This work demonstrated that the developed online, two-dimensional TFC/LC-MS method was effective in discovering the binding affinity of new compounds from natural extracts for target proteins, even at low concentrations.

  12. Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography

    PubMed Central

    Kanakaraj, Indhu; Jewell, David L.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2011-01-01

    Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and “histidine tags” genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu2+-iminodiacetic acid (IDA) agarose spin column, 94–99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu2+-IDA agarose) can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs. PMID:21264292

  13. Affinity chromatography of Drosophila melanogaster ribosomal proteins to 5S rRNA.

    PubMed

    Stark, B C; Chooi, W Y

    1985-02-20

    The binding of Drosophila melanogaster ribosomal proteins to D. melanogaster 5S rRNA was studied using affinity chromatography of total ribosomal proteins (TP80) on 5S rRNA linked via adipic acid dihydrazide to Sepharose 4B. Ribosomal proteins which bound 5S rRNA at 0.3 M potassium chloride and were eluted at 1 M potassium chloride were identified as proteins 1, L4, 2/3, L14/L16, and S1, S2, S3, S4, S5, by two-dimensional polyacrylamide gel electrophoresis. Using poly A-Sepharose 4B columns as a model of non-specific binding, we found that a subset of TP80 proteins is also bound. This subset, while containing some of the proteins bound by 5S rRNA columns, was distinctly different from the latter subset, indicating that the binding to 5S rRNA was specific for that RNA species. PMID:3923010

  14. MEASUREMENT OF DRUG-PROTEIN DISSOCIATION RATES BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PEAK PROFILING

    PubMed Central

    Schiel, John E.; Ohnmacht, Corey M.; Hage, David S.

    2012-01-01

    The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus non-retained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (± 0.2) s−1 was obtained by this approach at pH 7.4 and 37°C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates. PMID:19422253

  15. Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.

    PubMed

    Yuan, Jie; Zhou, Huifang; Yang, Yicong; Li, Weimin; Wan, Yi; Wang, Lili

    2015-05-01

    Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC.

  16. Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

    PubMed

    Ball, D J; Nishimura, J S

    1980-11-25

    Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits. PMID:7430155

  17. A new method of quantitative affinity chromatography and its application to the study of myosin.

    PubMed Central

    Bottomley, R C; Storer, A C; Trayer, I P

    1976-01-01

    A new method of quantifying the interactions between two or three components of an interacting system, one of which is insoluble, is described. The method differs from those previously applied to affinity chromatography systems in that it does not require that elution volumes be measured, but is instead dependent on measurements of the quantity of affinity-bound material. Theoretical expressions are derived for systems in which the acceptor is immobilized. Examples presented to illustrate the validity of the theory are of the latter type and are from studies on the myosin-adenosine nucleotide-PPi system. With Sepharose-myosin columns (myosin covalently coupled to CNBr-activated Sepharose) a dissociation constant of 1.8 muM for ATP4- was found. Data were also obtained under conditions that closely approximate to those found in vivo, i.e. on columns packed with a slurry of Sephadex G-50 and precipitated myosin filaments formed at low ionic strength. The binding of MgATP2-, MgADP-, ATP4- and MgPPi2- to "filamentous" myosin in both two- (myosin and nucleotide) and three- (myosin, nucleotide and PPi) component systems at different temperatures was studied and the dissociation constants obtained agreed well with previously published values. Except for the binding of ATP4- to filamentous myosin at 4 degrees when 85% of the protein was interacting with the nucleotide, much lower values for the number of available sites occupied by the nucleotides were as a routine found in this system. Although this apparent discrepancy is difficult to explain, it is not an anomaly of the theoretical approach and may reflect the present state of understanding of the myosin system. PMID:1008824

  18. Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

    PubMed Central

    Clarke, C M; Knowles, J R

    1977-01-01

    The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417]. PMID:23108

  19. CHARACTERIZATION OF THE BINDING OF SULFONYLUREA DRUGS TO HSA BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Joseph, K.S.; Hage, David S.

    2010-01-01

    Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (± 0.2) × 105 M−1 and 3.5 (± 3.0) × 102 M−1 for acetohexamide and values of 8.7 (± 0.6) × 104 and 8.1 (± 1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (± 0.1) × 105 and 4.3 (± 0.3) × 104 M−1, respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (± 0.2) × 104 and 5.3 (± 0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions. PMID:20435530

  20. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    PubMed

    Salehi, Nasrin; Peng, Ching-An

    2016-07-01

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. PMID:27110670

  1. Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.

    PubMed

    Yatsukawa, Yoh-Ichi; Ito, Hironobu; Matsuda, Takahiro; Nakamura, Munetomo; Watai, Masatoshi; Fujita, Kazuhiro

    2011-01-01

    A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.

  2. [Preparation and evaluation of pepsin affinity organic polymer capillary monolithic column].

    PubMed

    Chi, Cuijie; Wang, Wei; Ji, Yibing

    2014-08-01

    The protein modified monolithic column in affinity capillary electrochromatography (CEC) has attracted considerable attention over the past decades because of its great enantioseparation ability. A porous polymethacrylate ester-based capillary monolithic column poly (glycidyl methacrylate-co-ethyleneglycol dimethacrylate) (poly (GMA-co-EDMA)) was prepared by in situ co-polymerization. The process was initiated thermally by azobisisobutyronitrile (AIBN). The polymerization mixture contained GMA as the function monomer and EDMA as the crosslinking agent with 1,4-butanediol and 1-propanol as the binary porogen solvent. Under the optimized reaction conditions, including the proportion of monomer and porogens, reaction temperature etc, the column exhibited a uniform structure, sufficient permeability and excel- lent pressure resistance. The separation of alkyl benzenes on the column was mainly based on typical reversed-phase chromatographic retention mechanism. The reproducibility and stability were good with RSDs less than 9. 0%. A pepsin functionalized organic polymer monolith was prepared by covalently bonded pepsin to poly(GMA-co-EDMA) monolith with glutaraldehyde as a spacer based on the activity of epoxide group. The enantioseparation performance of the pepsin affinity monolith for basic enantiomers has been investigated by CEC. Nefopam, amlodipine, citalopram and chlorpheniramine were resolved, and baseline separations of nefopam, amlodipine, citalopram were achieved. The influences of pH, operating voltage, temperature and sample quantity used on the chiral separation were studied. The chiral recognition mechanism of enantiomers on the monolithic column in CEC is discussed. This work developed a new method for the prepataion and application of protein affinity monolith in CEC.

  3. Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-Layer Chromatography

    ERIC Educational Resources Information Center

    Davies, Don R.; Johnson, Todd M.

    2007-01-01

    A simple experiment for undergraduate organic chemistry students to separate a colorless mixture using column chromatography and then monitor the outcome of the separation using thin-layer chromatography (TLC) and infrared spectroscopy(IR) is described. The experiment teaches students the principle and techniques of column and thin-layer…

  4. Column liquid chromatography-ultraviolet and column liquid chromatography/mass spectrometry evaluation of stress degradation behavior of escitalopram oxalate.

    PubMed

    Dhaneshwar, Sunil R; Mahadik, Mahadeo V; Kulkarni, Mahesh J

    2009-01-01

    The objective of this work was to study the degradation behavior of escitalopram oxalate under different International Conference on Harmonization (ICH)-recommended stress conditions by column liquid chromatography (LC)-UV and LC/mass spectrometry (LC/MS) and to establish a validated stability-indicating LC assay method. Escitalopram oxalate was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal decomposition. Extensive degradation was found to occur in alkaline medium. Mild degradation was observed in acidic and oxidative conditions. Escitalopram oxalate was stable to neutral, photolytic, and thermal stress. Successful separation of the drug from degradation products formed under stress conditions was achieved on a PerfectSil-100 ODS-3 column [C18 (5 microm, 25 cm x 4.6 mm id)] using methanol-0.01 M acetate buffer pH 3.8 adjusted with acetic acid (45 + 55) as the mobile phase. The flow rate was 1 ml/min, and the detection wavelength was 239 nm. The method was validated according to ICH guidelines. Major degradation products formed in hydrolysis and oxidative conditions were isolated, and structural elucidation of degradation products was done by LCIMS and infrared spectrometry studies. The major hydrolysis degradation product was confirmed as 1-(3-dimethylaminopropyl)-1-(4-fluoro- phenyl)-1,3dihydroisobenzofuran-5-carboxylic acid, and the major oxidative degradation product was confirmed as 1-{[3-dimethylamino(oxide)- propyl]-1-(4-fluro-phenyl)}-1,3-dihydro-isobenzofuran- 5-carbonitrile.

  5. 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

  6. Frontal affinity chromatography: a unique research tool for biospecific interaction that promotes glycobiology.

    PubMed

    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

  7. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples. PMID:27498022

  8. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

  9. Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

    PubMed

    Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

    2015-01-01

    Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels. PMID:25749956

  10. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies.

    PubMed

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  11. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies

    PubMed Central

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  12. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  13. Analysis of global components in Ganoderma using liquid chromatography system with multiple columns and detectors.

    PubMed

    Qian, Zhengming; Zhao, Jing; Li, Deqiang; Hu, Dejun; Li, Shaoping

    2012-10-01

    In present study, a multiple columns and detectors liquid chromatography system for analysis of global components in traditional Chinese medicines was developed. The liquid chromatography system was consist of three columns, including size exclusion chromatography column, hydrophilic interaction chromatography column, and reversed phase chromatography column, and three detectors, such as diode array detector, evaporative light scattering detector, and mass spectrometry detector, based on column switching technique. The developed multiple columns and detectors liquid chromatography system was successfully applied to the analysis of global components, including macromolecular (polysaccharides), high (nucleosides and sugars)-, and low (triterpenes)-polarity small molecular compounds in Ganoderma, a well-known Chinese medicinal mushroom. As a result, one macromolecular chromatographic peak was found in two Ganoderma species, 19 components were identified in Ganoderma lucidum (two sugars, three nucleosides, and 14 triterpenes), and four components (two sugars and two nucleosides) were identified in Ganoderma sinense. The developed multiple columns and detectors liquid chromatography system was helpful to understand comprehensive chemical characters in TCMs.

  14. Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

    PubMed

    Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

    2016-05-15

    A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

  15. Purification of phage display-modified bacteriophage T4 by affinity chromatography

    PubMed Central

    2011-01-01

    Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be

  16. 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

  17. Analysis of Drug Interactions with Lipoproteins by High-Performance Affinity Chromatography

    PubMed Central

    Sobansky, Matthew R.; Hage, David S.

    2013-01-01

    Lipoproteins such as high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are known to interact with drugs and other solutes in blood. These interactions have been examined in the past by methods such as equilibrium dialysis and capillary electrophoresis. This chapter describes an alternative approach that has recently been developed for examining these interactions by using high-performance affinity chromatography. In this method, lipoproteins are covalently immobilized to a solid support and used within a column as a stationary phase for binding studies. This approach allows the same lipoprotein preparation to be used for a large number of binding studies, leading to precise estimates of binding parameters. This chapter will discuss how this technique can be applied to the identification of interaction models and be used to differentiate between systems that have interactions based on partitioning, adsorption or mixed-mode interactions. It is also shown how this approach can then be used for the measurement of binding parameters for HDL and LDL with drugs. Examples of these studies are provided, with particular attention being given to the use of frontal analysis to examine the interactions of R- and S-propranolol with HDL and LDL. The advantages and possible limitations of this method are described. The extension of this approach to other types of drug-lipoprotein interactions is also considered. PMID:25392741

  18. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

    PubMed

    Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S

    2016-01-01

    Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924

  19. BIOINTERACTION ANALYSIS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: KINETIC STUDIES OF IMMOBILIZED ANTIBODIES

    PubMed Central

    Nelson, Mary Anne; Moser, Annette; Hage, David S.

    2009-01-01

    A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody-antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7–12 × 106 M−1 at pH 7.0 and 25°C. Split-peak analysis gave association rate constants of 1.4–12 × 105 M−1s−1 and calculated dissociation rate constants of 0.01–0.4 s−1 under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056–0.17 s−1. A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4 × 10−4 s−1. This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports. PMID:19394281

  20. Cellufine sulfate column chromatography as a simple, rapid, and effective method to purify dengue virus.

    PubMed

    Kanlaya, Rattiyaporn; Thongboonkerd, Visith

    2016-08-01

    Conventional method to purify/concentrate dengue virus (DENV) is time-consuming with low virus recovery yield. Herein, we applied cellufine sulfate column chromatography to purify/concentrate DENV based on the mimicry between heparan sulfate and DENV envelope protein. Comparative analysis demonstrated that this new method offered higher purity (as determined by less contamination of bovine serum albumin) and recovery yield (as determined by greater infectivity). Moreover, overall duration used for cellufine sulfate column chromatography to purify/concentrate DENV was approximately 1/20 of that of conventional method. Therefore, cellufine sulfate column chromatography serves as a simple, rapid, and effective alternative method for DENV purification/concentration.

  1. Negative Enrichment of Target Cells by Microfluidic Affinity Chromatography

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2011-01-01

    A three-dimensional microfluidic channel was developed for high purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of non-target cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled, and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multi-step processes in microfluidic systems. In previous work, we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity, ease of fabrication and use, is suitable for cell separations when subsequent analysis of target cells is required. PMID:21939198

  2. Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC).

    PubMed

    de Costa, Fernanda; Barber, Carla J S; Pujara, Pareshkumar T; Reed, Darwin W; Covello, Patrick S

    2016-01-01

    Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography. PMID:26843168

  3. Dimerization Capacities of FGF2 Purified with or without Heparin-Affinity Chromatography

    PubMed Central

    Chiu, Liang-Yuan; Taouji, Said; Moroni, Elisabetta; Colombo, Giorgio; Chevet, Eric; Sue, Shih-Che; Bikfalvi, Andreas

    2014-01-01

    Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well. PMID:25299071

  4. Mixed-bed affinity chromatography: principles and methods.

    PubMed

    Boschetti, Egisto; Righetti, Pier Giorgio

    2015-01-01

    Mixed-bed chromatography is far from being a well-established technology within the panoply of bioseparation tools. Composed of an assembly of distinct sorbents that are mixed in a single bed, they have been mostly developed in the last decade for the reduction of dynamic concentration range where they allowed discovering many low-copy proteins within very complex proteomes. Other interesting preparative applications of mixed-bed chromatography have since been developed. In this chapter the basic concepts first and then detailed application recipes are described for (1) the reduction of protein dynamic concentration range, (2) the removal of impurity traces at the last stage of a biopurification process, and (3) the selection and use of sorbents as mixed bed in protein purification. PMID:25749952

  5. 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.

  6. Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

    PubMed

    O'Shaughnessy, Colette M; Micoli, Francesca; Gavini, Massimiliano; Goodall, Margaret; Cobbold, Mark; Saul, Allan; Maclennan, Calman A

    2013-01-31

    Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella

  7. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media.

  8. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media. PMID:27524303

  9. Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose.

    PubMed

    Larson, T J; Hirabayshi, T; Dowhan, W

    1976-03-01

    Cytidinediphospho-sn-1,2-diaclglycerol (CDP-diglyceride) has been covalently linked to Sephrose 4B via adipic acid dihydrazide spacer arm forming an effective affinity chromatography column. This liponucleo-tide ligand and sn-glycero-3-phosphate are subtracts for the formation of 3-sn-phoshatidyl-1'-sn-glycero-3'-phosphate (PGP) catalyzed in both eukaryotic and prokaryotic organisms by sn-glycero-3-phosphate: CMP phosphatidlytranferase (PGP synthetase). Using this CDP-diglyceride Sephrose affinity column we were able to resolve the membrane associated 3-sn-phosphatidyl'1-sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities. A PGP synthetase activity was adsorbed to the affinity column and was eluted using buffer containg CDP-diglyceride; a PGP phosphatease acactivity had no affinity for the column. Both PGP synthase and PGP phosphatase of B. licheniformis were associated with a membrane component of the cell as evidenced by sucrose gradient centrifugation, differential centrifugation, and solubilization by buffers containing detergent... PMID:175832

  10. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  11. Glycan-specific whole cell affinity chromatography: a versatile microbial adhesion platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have constructed a C-glycoside ketohydrazide affinity chromatography resin that interacts with viable whole-cell microbial populations with biologically appropriate stereo-specificity in a carbohydrate-defined manner. It readily allows for the quantification, selection, and manipulation of target...

  12. Selective retention of basic compounds by metal aquo-ion affinity chromatography.

    PubMed

    Asakawa, Yoshiki; Yamamoto, Eiichi; Asakawa, Naoki

    2014-10-01

    A novel metal aquo-ion affinity chromatography has been developed for the analysis of basic compounds using heat-treated silica gel containing hydrated metal cations (metal aquo-ions) as the packing material. The packing materials of the metal aquo-ion affinity chromatography were prepared by the immobilization of a single metal component such as Fe(III), Al(III), Ag(I), and Ni(II) on silica gel followed by extensive heat treatment. The immobilized metals form aquo-ions to present cation-exchange ability for basic analytes and the cation-exchange ability for basic analytes depends on pKa of the immobilized metal species. In the present study, to evaluate the retention characteristics of metal aquo-ion affinity chromatography, the on-line solid-phase extraction of drugs was investigated. Obtained data clearly evidence the selective retention capability of metal aquo-ion affinity chromatography for basic analytes with sufficient capacity. PMID:25044622

  13. NON-COMPETITIVE PEAK DECAY ANALYSIS OF DRUG-PROTEIN DISSOCIATION BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Jianzhong; Schiel, John E.; Hage, David S.

    2009-01-01

    The peak decay method is an affinity chromatographic technique that has been used to examine the dissociation of solutes from immobilized ligands in the presence of excess displacing agent. However, it can be difficult to find a displacing agent that does not interfere with detection of the eluting analyte. In this study, a non-competitive peak decay method was developed in which no displacing agent was required for analyte elution. This method was evaluated for the study of drug-protein interactions by using it along with high-performance affinity chromatography to measure the dissociation rate constants for R- and S-warfarin from columns containing immobilized human serum albumin (HSA). Several factors were considered in the optimization of this method, including the amount of applied analyte, the column size, and the flow rate. The dissociation rate constants for R- and S-warfarin from HSA were measured at several temperatures by this approach, giving values of 0.56 (± 0.01) and 0.66 (± 0.01) s−1 at pH 7.4 and 37°C. These results were in good agreement with previous values obtained by other methods. This approach is not limited to warfarin and HSA but could be employed in studying additional drug-protein interactions or other systems with weak-to-moderate binding. PMID:19472288

  14. Separation of hydrophobic metabolites using monolithic silica column in high-performance liquid chromatography and supercritical fluid chromatography.

    PubMed

    Bamba, Takeshi; Fukusaki, Eiichiro

    2009-08-01

    Monolithic silica columns have very low back-pressures and offer several advantages over conventional columns packed with spherical particles, such as high separation efficiency and rapid analysis. In this review, we report the applicability of monolithic silica columns for the analysis of complex hydrophobic metabolites. We have used monolithic columns in HPLC and developed a separation technique for the high-speed and high-resolution analysis of the geometric analogs of natural polyprenols. We also used monolithic columns in supercritical fluid chromatography for the successful separation of the structural isomers of carotenoids after deciding the analytical conditions that were suitable for this separation and have developed a method for profiling biological samples containing complex matrices. We have proved that excellent resolution can be obtained by connecting a number of monolithic columns in series.

  15. Colorful Column Chromatography: A Classroom Demonstration of a Three-Component Separation

    ERIC Educational Resources Information Center

    Heumann, Lars V.

    2008-01-01

    A classroom demonstration detailing the procedure for the separation of a ternary mixture consisting of intensely colored compounds using silica gel column chromatography is described. The audience can follow the compounds during their passage through the column as individual, colored bands while learning about different tools and techniques used…

  16. 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.

  17. The quest for affinity chromatography ligands: are the molecular libraries the right source?

    PubMed

    Perret, Gérald; Santambien, Patrick; Boschetti, Egisto

    2015-08-01

    Affinity chromatography separations of proteins call for highly specific ligands. Antibodies are the most obvious approach; however, except for specific situations, technical and economic reasons are arguments against this choice especially for preparative purposes. With this in mind, the rationale is to select the most appropriate ligands from collections of pre-established molecules. To reach the objective of having a large structural coverage, combinatorial libraries have been proposed. These are classified according to their nature and origin. This review presents and discusses the most common affinity ligand libraries along with the most appropriate screening methods for the identification of the right affinity chromatography selective structure according to the type of library; a side-by-side comparison is also presented. PMID:26033846

  18. Optimization of human serum albumin monoliths for chiral separations and high-performance affinity chromatography

    PubMed Central

    Pfaunmiller, Erika L.; Hartmann, Mahli; Dupper, Courtney M.; Soman, Sony; Hage, David S.

    2012-01-01

    Various organic-based monoliths were prepared and optimized for immobilization of the protein human serum albumin (HSA) as a binding agent for chiral separations and high-performance affinity chromatography. These monoliths contained co-polymers based on glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or GMA and trimethylolpropane trimethacrylate (TRIM). A mixture of cyclohexanol and 1-dodecanol was used as the porogen, with the ratio of these solvents being varied along with the polymerization temperature to generate a library of monoliths. These monoliths were used with both the Schiff base and epoxy immobilization methods and measured for their final content of HSA. Monoliths showing the highest protein content were further evaluated in chromatographic studies using R/S-warfarin and d/l-tryptophan as model chiral solutes. A 2.6–2.7-fold increase in HSA content was obtained in the final monoliths when compared to similar HSA monoliths prepared according to the literature. The increased protein content made it possible for the new monoliths to provide higher retention and/or two-fold faster separations for the tested solutes when using 4.6 mm i.d. × 50 mm columns. These monoliths were also used to create 4.6 mm i.d. × 10 mm HSA microcolumns that could separate the same chiral solutes in only 1.5–6.0 min. The approaches used in this study could be extended to the separation of other chiral solutes and to the optimization of organic monoliths for use with additional proteins as binding agents. PMID:23010249

  19. 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

    2016-06-01

    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. PMID:27155298

  20. 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

    2016-06-01

    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.

  1. Polymeric Cryogel-Based Boronate Affinity Chromatography for Separation of Ribonucleic Acid from Bacterial Extracts.

    PubMed

    Shakya, Akhilesh Kumar; Srivastava, Akshay; Kumar, Ashok

    2015-01-01

    Three-dimensional monolithic columns are preferred stationary phase in column chromatography. Conventional columns based on silica or particles are efficient in bioseparation though associated with limitations of nonspecific interaction and uneven porosity that causes high mass transfer resistance for the movement of big molecules. Cryogels as a monolith column have shown promising application in bioseparation. Cryogels column can be synthesized in the form of a monolith at sub-zero temperature through gelation of pre-synthesized polymers or polymerization of monomers. Cryogels are macroporous and mechanically stable materials. They have open interconnected micron-sized pores with a wide range of porosity (10-200 μm). Current protocol demonstrated the ability of poly(hydroxymethyl methacrylate)-co-vinylphenyl boronic acid p(HEMA-co-VPBA) cryogel matrix for selective separation of RNA from the bacterial crude extract. PMID:26623972

  2. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate.

  3. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. PMID:26363185

  4. Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography.

    PubMed

    Nestola, Piergiuseppe; Silva, Ricardo J S; Peixoto, Cristina; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

    2014-06-20

    Adenovirus serotype 5 (Ad5) was successfully separated by size-exclusion chromatography (SEC) using a simple, yet efficient, two-column, quasi-continuous, simulated moving-bed process operated in an open-loop configuration. The operating cycle is divided into two identical half-cycles, each of them consisting of the following sequence of sub-steps: (i) elution of the upstream column and direction of the effluent of the downstream column to waste; (ii) elution of the upstream column and redirection of its effluent to waste while the downstream column is fed with the clarified bioreaction bulk and its effluent collected as purified product; (iii) operation of the system as in step (i) but collecting the effluent of the downstream column as product; (iv) elution of the upstream column and direction of its effluent to waste while the flow through the downstream column is temporarily halted. Clearance of impurities, namely DNA and host cell protein (HCP), were experimentally assessed. The pilot-scale run yielded a virus recovery of 86%, and a clearance of 90% and 89% for DNA and HCP, respectively, without any fine tunning of the predetermined operating parameters. These figures compare very favorably against single-column batch chromatography for the same volume of size-exclusion resin. However, and most importantly, the virus yield was increased from 57% for the batch system to 86% for the two-column SEC process because of internal recycling of the mixed fractions of contaminated Ad5, even though the two-column process was operated strictly in an open-loop configuration. And last, but not least, the productivity was increased by 6-fold with the two-column process. In conclusion, the main drawbacks of size-exclusion chromatography, namely low productivity and low product titer, were overcome to a considerable extent by an innovative two-column configuration that keeps the mixed fractions inside the system at all times.

  5. Evaluation of SDS depletion using an affinity spin column and IMS-MS detection

    SciTech Connect

    Hengel, Shawna M.; Floyd, Erica A.; Baker, Erin Shammel; Zhao, Rui; Wu, Si; Pasa-Tolic, Ljiljana

    2012-11-01

    While the use of detergents is necessary for a variety of protein isolation preparation protocols, often prior to mass spectral (MS) analysis, they are not compatible with MS analysis due to ion suppression and adduct formation. This manuscript describes optimization of detergent removal, using commercially available SDS depletion spin columns containing an affinity resin, providing for both increased protein recovery and thorough SDS removal. Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) allowed for a concurrent analysis of both analyte and detergent. In the case of both proteins and peptides, higher detergent concentrations than previously reported provided an increase of sample recovery; however there was a limit as SDS was detected by IMS-MS at higher levels of SDS indicating incomplete detergent depletion. The results also suggest optimal conditions for SDS removal are dependent on the sample concentration. Overall, this study provides a useful guide for proteomic studies where SDS is required for efficient sample preparation.

  6. Generator for ionic gallium-68 based on column chromatography

    DOEpatents

    Neirinckx, Rudi D.; Davis, Michael A.

    1981-01-01

    A physiologically acceptable solution of gallium-68 fluorides, having an activity of 0.1 to 50 millicuries per milliliter of solution is provided. The solution is obtained from a generator comprising germanium-68 hexafluoride bound to a column of an anion exchange resin which forms gallium-68 in situ by eluting the column with an acid solution to form a solution containing .sup.68 Ga-fluorides. The solution then is neutralized prior to administration.

  7. Preparation and Characterization of a Polymeric Monolithic Column for Use in High-Performance Liquid Chromatography (HPLC)

    ERIC Educational Resources Information Center

    Bindis, Michael P.; Bretz, Stacey Lowery; Danielson, Neil D.

    2011-01-01

    The high-performance liquid chromatography (HPLC) experiment, most often done in the undergraduate analytical instrumentation laboratory course, generally illustrates reversed-phase chromatography using a commercial C[subscript]18 silica column. To avoid the expense of periodic column replacement and introduce a choice of columns with different…

  8. 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.

  9. 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. PMID:26518492

  10. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

    PubMed

    Müller, Egbert; Vajda, Judith

    2016-05-15

    Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected. PMID:26830536

  11. 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. PMID:26228853

  12. 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.

  13. IDENTIFICATION AND ANALYSIS OF STEREOSELECTIVE DRUG INTERACTIONS WITH LOW DENSITY LIPOPROTEIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Sobansky, Matthew R.; Hage, David S.

    2012-01-01

    Columns containing immobilized low density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4, 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nKa) of 1.9 (± 0.1) × 105 M-1 for R-propranolol and 2.7 (± 0.2) × 105 M-1 for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (Ka) of 5.2 (± 2.3) × 105 M-1 at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents. PMID:22354572

  14. The derivatization of oxidized polysaccharides for protein immobilization and affinity chromatography.

    PubMed

    Junowicz, E; Charm, S E

    1976-03-25

    The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5'-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography. PMID:1260016

  15. A straightforward methodology for designing continuous monoclonal antibody capture multi-column chromatography processes.

    PubMed

    Gjoka, Xhorxhi; Rogler, Karl; Martino, Richard Alexander; Gantier, Rene; Schofield, Mark

    2015-10-16

    A simple process development strategy for continuous capture multi-column chromatography (MCC) is described. The approach involves a few single column breakthrough experiments, based on several simplifying observations that enable users to rapidly convert batch processes into well-designed multi-column processes. The method was validated using a BioSMB(®) (Pall Life Sciences) lab scale multi-column system and a mAb capture process employing Protein A resin. The approach enables users to optimize MCC processes based on their internal preferences and constraints without requiring any mathematical modeling expertise.

  16. Affinity chromatography of nicotinamide-adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide.

    PubMed

    Barry, S; O'Carra, P

    1973-12-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems

  17. Developing Inquiry-Based Labs Using Micro-Column Chromatography

    ERIC Educational Resources Information Center

    Barden-Gabbei, Laura M.; Moffitt, Deborah L.

    2006-01-01

    Chromatography is a process by which mixtures can be separated or substances can be purified. Biological and chemical laboratories use many different types of chromatographic processes. For example, the pharmaceutical industry uses chromatographic techniques to purify drugs, medical labs use them to identify blood components such as cholesterol,…

  18. Highly crosslinked silicon polymers for gas chromatography columns

    NASA Technical Reports Server (NTRS)

    Shen, Thomas C. (Inventor)

    1994-01-01

    A new highly crosslinked silicone polymer particle for gas chromatography application and a process for synthesizing such copolymer are described. The new copolymer comprises vinyltriethoxysilane and octadecyltrichlorosilane. The copolymer has a high degree of crosslinking and a cool balance of polar to nonpolar sites in the porous silicon polymer assuring fast separation of compounds of variable polarity.

  19. Specific recognition of supercoiled plasmid DNA by affinity chromatography using a synthetic aromatic ligand.

    PubMed

    Caramelo-Nunes, Catarina; Tomaz, Cândida T

    2015-01-01

    Liquid chromatography is the method of choice for the purification of plasmid DNA (pDNA), since it is simple, robust, versatile, and highly reproducible. The most important features of a chromatographic procedure are the use of suitable stationary phases and ligands. As conventional purification protocols are being replaced by more sophisticated and selective procedures, the focus changes toward designing and selecting ligands of high affinity and specificity. In fact, the chemical composition of the chromatographic supports determines the interactions established with the target molecules, allowing their preferential retention over the undesirable ones. Here it is described the selective recognition and purification of supercoiled pDNA by affinity chromatography, using an intercalative molecule (3,8-diamino-6-phenylphenanthridine) as ligand. PMID:25749945

  20. Miniaturized protein separation using a liquid chromatography column on a flexible substrate

    NASA Astrophysics Data System (ADS)

    Yang, Yongmo; Chae, Junseok

    2008-12-01

    We report a prototype protein separator that successfully miniaturizes existing technology for potential use in biocompatible health monitoring implants. The prototype is a liquid chromatography (LC) column (LC mini-column) fabricated on an inexpensive, flexible, biocompatible polydimethylsiloxane (PDMS) enclosure. The LC mini-column separates a mixture of proteins using size exclusion chromatography (SEC) with polydivinylbenzene beads (5-20 µm in diameter with 10 nm pore size). The LC mini-column is smaller than any commercially available LC column by a factor of ~11 000 and successfully separates denatured and native protein mixtures at ~71 psi of the applied fluidic pressure. Separated proteins are analyzed using NuPAGE-gel electrophoresis, high-performance liquid chromatography (HPLC) and an automated electrophoresis system. Quantitative HPLC results demonstrate successful separation based on intensity change: within 12 min, the intensity between large and small protein peaks changed by a factor of ~20. In further evaluation using the automated electrophoresis system, the plate height of the LC mini-column is between 36 µm and 100 µm. The prototype LC mini-column shows the potential for real-time health monitoring in applications that require inexpensive, flexible implant technology that can function effectively under non-laboratory conditions.

  1. Robust naphthyl methacrylate monolithic column for high performance liquid chromatography of a wide range of solutes.

    PubMed

    Jonnada, Murthy; El Rassi, Ziad

    2015-08-28

    An organic monolithic column based on the co-polymerization of 2-naphthyl methacrylate (NAPM) as the functional monomer and trimethylolpropane trimethacrylate (TRIM) as the crosslinker was introduced for high performance reversed-phase liquid chromatography (RPC). The co-polymerization was performed in situ in a stainless steel column of 4.6mm i.d. in the presence of a ternary porogen consisting of 1-dodecanol and cyclohexanol. This monolithic column (referred to as naphthyl methacrylate monolithic column or NMM column) showed high mechanical stability at relatively high mobile phase flow velocity indicating that the column has excellent hydrodynamic characteristics. To characterize the NMM column, different probe molecules including alkyl benzenes, and aniline, benzene, toluene and phenol derivatives were chromatographed on the column and the results in terms of k, selectivity and plate counts were compared to those obtained on an octadecyl silica (ODS) column in order to assess the presence of π-π and hydrophobic interactions on the NMM column under otherwise the same elution conditions. The NMM column offered additional π-π interactions with aromatic molecules in addition to hydrophobic interactions under RPC elution conditions. Run-to-run and column-to-column reproducibility of solute k values were evaluated, and percent relative standard deviation of <1% and ∼2-3.5%, respectively, were obtained. Six standard proteins were readily separated on the NMM column using shallow (30min at 1.0mL/min), steep (10min at 1.0mL/min) and ultra steep (1min at 3.0mL/min) linear gradient elution at increasing ACN concentration in the mobile phase using a 10cm×4.6mm i.d. column in case of shallow and steep linear gradients and a 3cm×4.6mm i.d. column for ultra steep linear gradient.

  2. How to select equivalent and complimentary reversed phase liquid chromatography columns from column characterization databases.

    PubMed

    Borges, Endler M

    2014-01-01

    Three RP-LC column characterization protocols [Tanaka et al. (1989), Snyder et al. (PQRI, 2002), and NIST SRM 870 (2000)] were evaluated using both Euclidian distance and Principal Components Analysis to evaluate effectiveness at identifying equivalent columns. These databases utilize specific chromatographic properties such as hydrophobicity, hydrogen bonding, shape/steric selectivity, and ion exchange capacity of stationary phases. The chromatographic parameters of each test were shown to be uncorrelated. Despite this, the three protocols were equally successful in identifying similar and/or dissimilar stationary phases. The veracity of the results has been supported by some real life pharmaceutical separations. The use of Principal Component Analysis to identify similar/dissimilar phases appears to have some limitations in terms of loss of information. In contrast, the use of Euclidian distances is a much more convenient and reliable approach. The use of auto scaled data is favoured over the use of weighted factors as the former data transformation is less affected by the addition or removal of columns from the database. The use of these free databases and their corresponding software tools shown to be valid for identifying similar columns with equivalent chromatographic selectivity and retention as a "backup column". In addition, dissimilar columns with complimentary chromatographic selectivity can be identified for method development screening strategies.

  3. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

    PubMed

    Jia, Yinshan; Jarrett, Harry W

    2015-08-01

    The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach. PMID:25935261

  4. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose

    PubMed Central

    Jia, Yinshan; Jarrett, Harry W.

    2015-01-01

    The uses of a method of coupling DNA is investigated for trapping and purifying transcription factors. Using the GFP-C/EBP fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry utilized is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA-binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose which couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes including E2A, c-myc, and myo-D were also purified but myogenenin and NFκB were not. Therfore, this approach proved valuable for both affinity chromatography and for the trapping approach. PMID:25935261

  5. A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

    PubMed Central

    Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2013-01-01

    Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

  6. Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Voráčková, Irena; Suchanová, Šárka; Ulbrich, Pavel; Diehl, William E.; Ruml, Tomáš

    2011-01-01

    Heterologous proteins are frequently purified by Immobilized Metal Ion Affinity Chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e. CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state. PMID:21600288

  7. Protecting group-free immobilization of glycans for affinity chromatography using glycosylsulfonohydrazide donors.

    PubMed

    Hernandez Armada, Daniel; Santos, Jobette T; Richards, Michele R; Cairo, Christopher W

    2015-11-19

    A variety of applications in glycobiology exploit affinity chromatography through the immobilization of glycans to a solid support. Although several strategies are known, they may provide certain advantages or disadvantages in how the sugar is attached to the affinity matrix. Additionally, the products of some methods may be hard to characterize chemically due to non-specific reactions. The lack of specificity in standard immobilization reactions makes affinity chromatography with expensive oligosaccharides challenging. As a result, methods for specific and efficient immobilization of oligosaccharides remain of interest. Herein, we present a method for the immobilization of saccharides using N'-glycosylsulfonohydrazide (GSH) carbohydrate donors. We have compared GSH immobilization to known strategies, including the use of divinyl sulfone (DVS) and cyanuric chloride (CC), for the generation of affinity matrices. We compared immobilization methods by determining their immobilization efficiency, based on a comparison of the mass of immobilized carbohydrate and the concentration of active binding sites (determined using lectins). Our results indicate that immobilization using GSH donors can provide comparable amounts of carbohydrate epitopes on solid support while consuming almost half of the material required for DVS immobilization. The lectin binding capacity observed for these two methods suggests that GSH immobilization is more efficient. We propose that this method of oligosaccharide immobilization will be an important tool for glycobiologists working with precious glycan samples purified from biological sources. PMID:26454791

  8. Analyzing insulin samples by size-exclusion chromatography: a column degradation study.

    PubMed

    Teska, Brandon M; Kumar, Amit; Carpenter, John F; Wempe, Michael F

    2015-04-01

    Investigating insulin analogs and probing their intrinsic stability at physiological temperature, we observed significant degradation in the size-exclusion chromatography (SEC) signal over a moderate number of insulin sample injections, which generated concerns about the quality of the separations. Therefore, our research goal was to identify the cause(s) for the observed signal degradation and attempt to mitigate the degradation in order to extend SEC column lifespan. In these studies, we used multiangle light scattering, nuclear magnetic resonance, and gas chromatography-mass spectrometry methods to evaluate column degradation. The results from these studies illustrate: (1) that zinc ions introduced by the insulin product produced the observed column performance issues; and (2) that including ethylenediaminetetraacetic acid, a zinc chelator, in the mobile phase helped to maintain column performance.

  9. 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.

  10. Affinity chromatography purification of angiotensin II reactor using photoactivable biotinylated probes

    SciTech Connect

    Marie, J.; Seyer, R.; Lombard, C.; Desarnaud, F.; Aumelas, A.; Jard, A.; Bonnafous, J.C. )

    1990-09-25

    The authors have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH{sub 2}){sub 2}SS(CH{sub 2}){sub 2}CO-(Ala{sup 1}, Phe(4N{sub 3}){sup 8})AII, which contains a cleavage disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (K{sub d}{approximately}1 nM), proved to be suitable for indirect affinity chromatography of rate liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

  11. Structural assessment of beta-glucuronidase carbohydrate chains by lectin affinity chromatography.

    PubMed

    Wójczyk, B; Hoja, D; Lityńska, A

    1993-04-01

    Rat liver beta-glucuronidase was studied by sequential lectin affinity chromatography. beta-Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography. Ulex europaeus agglutinin-agarose chromatography revealed the presence of alpha(1-3)linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin, Ricinus communis agglutinin and Phaseolus vulgaris erythroagglutinin. PMID:8400827

  12. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.

  13. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. PMID:25277090

  14. High performance aptamer affinity chromatography for single-step selective extraction and screening of basic protein lysozyme.

    PubMed

    Han, Bin; Zhao, Chao; Yin, Junfa; Wang, Hailin

    2012-08-15

    A DNA aptamer based high-performance affinity chromatography is developed for selective extraction and screening of a basic protein lysozyme. First, a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic column was synthesized in situ by thermally initiated radical polymerization, and then an anti-lysozyme DNA aptamer was covalently immobilized on the surface of the monolith through a 16-atom spacer arm. The target protein lysozyme but non-target proteins can be trapped by the immobilized anti-lysozyme DNA aptamer. In contrast, lysozyme cannot be trapped by the immobilized oligodeoxynucleotide that does not contain the sequence of the anti-lysozyme DNA aptamer. The study clearly demonstrates the trapping of lysozyme by the immobilized anti-lysozyme DNA aptamer is mainly due to specific recognition rather than simple electrostatic interaction of positively charged protein and the negatively charged DNA. The inter-day precision was determined as 0.8% for migration time and 4.2% for peak area, respectively. By the use of aptamer affinity monolith, a screening strategy is developed to selectively extract lysozyme from chicken egg white, showing the advantages of high efficiency, low cost and ease-of-operation.

  15. Chemometrics applications in biotechnology processes: predicting column integrity and impurity clearance during reuse of chromatography resin.

    PubMed

    Rathore, Anurag S; Mittal, Shachi; Lute, Scott; Brorson, Kurt

    2012-01-01

    Separation media, in particular chromatography media, is typically one of the major contributors to the cost of goods for production of a biotechnology therapeutic. To be cost-effective, it is industry practice that media be reused over several cycles before being discarded. The traditional approach for estimating the number of cycles a particular media can be reused for involves performing laboratory scale experiments that monitor column performance and carryover. This dataset is then used to predict the number of cycles the media can be used at manufacturing scale (concurrent validation). Although, well accepted and widely practiced, there are challenges associated with extrapolating the laboratory scale data to manufacturing scale due to differences that may exist across scales. Factors that may be different include: level of impurities in the feed material, lot to lot variability in feedstock impurities, design of the column housing unit with respect to cleanability, and homogeneity of the column packing. In view of these challenges, there is a need for approaches that may be able to predict column underperformance at the manufacturing scale over the product lifecycle. In case such an underperformance is predicted, the operators can unpack and repack the chromatography column beforehand and thus avoid batch loss. Chemometrics offers one such solution. In this article, we present an application of chemometrics toward the analysis of a set of chromatography profiles with the intention of predicting the various events of column underperformance including the backpressure buildup and inefficient deoxyribonucleic acid clearance.

  16. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  17. Improved micromachined column design and fluidic interconnects for programmed high-temperature gas chromatography separations.

    PubMed

    Gaddes, David; Westland, Jessica; Dorman, Frank L; Tadigadapa, Srinivas

    2014-07-01

    This work focuses on the development and experimental evaluation of micromachined chromatographic columns for use in a commercial gas chromatography (GC) system. A vespel/graphite ferrule based compression sealing technique is presented using which leak-proof fluidic interconnection between the inlet tubing and the microchannel was achieved. This sealing technique enabled separation at temperatures up to 350°C on a μGC column. This paper reports the first high-temperature separations in microfabricated chromatographic columns at these temperatures. A 2m microfabricated column using a double Archimedean spiral design with a square cross-section of 100μm×100μm has been developed using silicon microfabrication techniques. The microfabricated column was benchmarked against a 2m 100μm diameter commercial column and the performance between the two columns was evaluated in tests performed under identical conditions. High temperature separations of simulated distillation (ASTM2887) and polycyclic aromatic hydrocarbons (EPA8310) were performed using the μGC column in temperature programmed mode. The demonstrated μGC column along with the high temperature fixture offers one more solution toward potentially realizing a portable μGC device for the detection of semi-volatile environmental pollutants and explosives without the thermal limitations reported to date with μGC columns using epoxy based interconnect technology. PMID:24866564

  18. Improved micromachined column design and fluidic interconnects for programmed high-temperature gas chromatography separations.

    PubMed

    Gaddes, David; Westland, Jessica; Dorman, Frank L; Tadigadapa, Srinivas

    2014-07-01

    This work focuses on the development and experimental evaluation of micromachined chromatographic columns for use in a commercial gas chromatography (GC) system. A vespel/graphite ferrule based compression sealing technique is presented using which leak-proof fluidic interconnection between the inlet tubing and the microchannel was achieved. This sealing technique enabled separation at temperatures up to 350°C on a μGC column. This paper reports the first high-temperature separations in microfabricated chromatographic columns at these temperatures. A 2m microfabricated column using a double Archimedean spiral design with a square cross-section of 100μm×100μm has been developed using silicon microfabrication techniques. The microfabricated column was benchmarked against a 2m 100μm diameter commercial column and the performance between the two columns was evaluated in tests performed under identical conditions. High temperature separations of simulated distillation (ASTM2887) and polycyclic aromatic hydrocarbons (EPA8310) were performed using the μGC column in temperature programmed mode. The demonstrated μGC column along with the high temperature fixture offers one more solution toward potentially realizing a portable μGC device for the detection of semi-volatile environmental pollutants and explosives without the thermal limitations reported to date with μGC columns using epoxy based interconnect technology.

  19. [Application of electroosmotic pump on micro column liquid chromatography].

    PubMed

    Chen, Ling-Xin; Guan, Ya-Feng

    2002-03-01

    An electroosmotic pump(EOP) was designed and evaluated, which could replace the mechanical pump. The EOP could generate 2.0 MPa-6.0 MPa output pressure and tens of nL/min-3 microL/min flow rate. A test mixture containing naphthalene, anthracene and phenanthrene was separated on a 14 cm x 320 microns i.d., 5 microns, C18 micro column with acetonitrile/water as a mobile phase, which demonstrated the applicability of the EOP.

  20. Biotin-functionalized poly(ethylene terephthalate) capillary-channeled polymer fibers as HPLC stationary phase for affinity chromatography.

    PubMed

    Jiang, Liuwei; Marcus, R Kenneth

    2015-01-01

    Native poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers have been used as the stationary phase for high-performance liquid chromatography (HPLC) of proteins via reversed-phase and ion-exchange processes. Functionalization can be used to bring about greater selectivity through surface modification. PET fibers were treated with ethylenediamine to generate primary amine groups on the fiber surface, enabling subsequent covalent attachment of ligands. The ninhydrin test for primary amines revealed surface densities of 13.9-60.0 μmol m(-2) for PET fibers exposed for periods of 3-12 min. Here, 8-amino-3,6-dioxaoctanoic acid was linked to the EDA-treated PET fiber surface as a hydrophilic spacer, and then D-biotin was attached on the end of the spacer as an affinity ligand. The streptavidin binding capacity and binding homogeneity were studied on the biotin-functionalized PET C-CP fiber microbore column. The selectivity of the biotin surface functionalization was assessed by spiking lysate with Texas Red-labeled streptavidin and enhanced green fluorescent protein. Greater than 99% selectivity was realized. This ligand-coupling strategy from standard solid-phase peptide synthesis used in stationary phase functionalization creates great potential for PET C-CP fiber-packed HPLC columns to perform a variety of chromatographic separations. PMID:25410640

  1. Optimization of immobilized gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests.

    PubMed

    Aryal, Uma K; Olson, Douglas J H; Ross, Andrew R S

    2008-12-01

    Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides. PMID:19183793

  2. ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: BINDING OF GLIBENCLAMIDE TO NORMAL AND GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2012-01-01

    High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, Ka, 1.4–1.9 × 106 M−1 at pH 7.4 and 37°C) and lower affinity sites (Ka, 4.4–7.2 × 104 M−1). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood. PMID:23092871

  3. Sub-to super-ambient temperature programmable microfabricated gas chromatography column

    DOEpatents

    Robinson, Alex L.; Anderson, Lawrence F.

    2004-03-16

    A sub- to super-ambient temperature programmable microfabricated gas chromatography column enables more efficient chemical separation of chemical analytes in a gas mixture by combining a thermoelectric cooler and temperature sensing on the microfabricated column. Sub-ambient temperature programming enables the efficient separation of volatile organic compounds and super-ambient temperature programming enables the elution of less volatile analytes within a reasonable time. The small heat capacity and thermal isolation of the microfabricated column improves the thermal time response and power consumption, both important factors for portable microanalytical systems.

  4. One-pot preparation of a sulfamethoxazole functionalized affinity monolithic column for selective isolation and purification of trypsin.

    PubMed

    Xiao, Yuan; Guo, Jialiang; Ran, Danni; Duan, Qianqian; Crommen, Jacques; Jiang, Zhengjin

    2015-06-26

    A facile and efficient "one-pot" copolymerization strategy was used for the preparation of sulfonamide drug (SA) functionalized monolithic columns. Two novel SA-immobilized methacrylate monolithic columns, i.e. poly(GMA-SMX-co-EDMA) and poly(GMA-SAA-co-EDMA) were prepared by one-pot in situ copolymerization of the drug ligand (sulfamethoxazole (SMX) or sulfanilamide (SAA)), the monomer (glycidyl methacrylate, GMA) and the cross-linker (ethylene dimethacrylate, EDMA) within 100 μm i.d. capillaries under optimized polymerization conditions. The physicochemical properties and column performance of the fabricated monolithic columns were characterized by elemental analysis, scanning electron microscopy and micro-HPLC. Satisfactory column permeability, efficiency and separation performance were obtained on the optimized poly(GMA-SMX-co-EDMA) monolithic column for small molecules, such as a standard test mixture and eight aromatic ketones. Notably, it was found that the poly(GMA-SMX-co-EDMA) monolith showed a selective affinity to trypsin, while the poly(GMA-SAA-co-EDMA) monolith containing sulfanilamide did not exhibit such affinity at all. This research not only provides a novel monolith for the selective isolation and purification of trypsin, but it also offers the possibility to easily prepare novel drug functionalized methacrylate monoliths through a one-pot copolymerization strategy.

  5. Effect of extra-column volume on practical chromatographic parameters of sub-2-μm particle-packed columns in ultra-high pressure liquid chromatography.

    PubMed

    Wu, Naijun; Bradley, Ashley C; Welch, Christopher J; Zhang, Li

    2012-08-01

    Effects of extra-column volume on apparent separation parameters were studied in ultra-high pressure liquid chromatography with columns and inlet connection tubings of various internal diameters (id) using 50-mm long columns packed with 1.8-μm particles under isocratic conditions. The results showed that apparent retention factors were on average 5, 11, 18, and 41% lower than those corrected with extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns, respectively, when the extra-column volume (11.3 μL) was kept constant. Also, apparent pressures were 31, 16, 12, and 10% higher than those corrected with pressures from extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns at the respective optimum flow rate for a typical ultra-high pressure liquid chromatography system. The loss in apparent efficiency increased dramatically from 4.6- to 3.0- to 2.1- to 1.0-mm id columns, less significantly as retention factors increased. The column efficiency was significantly improved as the inlet tubing id was decreased for a given column. The results suggest that maximum ratio of extra-column volume to column void volume should be approximately 1:10 for column porosity more than 0.6 and a retention factor more than 5, where 80% or higher of theoretically predicted efficiency could be achieved.

  6. Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

    PubMed

    Zhao, Wei-Wei; Liu, Fu-Feng; Shi, Qing-Hong; Sun, Yan

    2014-09-12

    In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations. PMID:25064536

  7. Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography

    SciTech Connect

    Jang, H.D.; Swaisgood, H.E. )

    1990-12-01

    Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.

  8. A mutant trypsin-like enzyme from Streptomyces fradiae, created by site-directed mutagenesis, improves affinity chromatography for protein trypsin inhibitors.

    PubMed

    Katoh, T; Kikuchi, N; Nagata, K; Yoshida, N

    1996-08-01

    The Ser-170 residue of a trypsin-like enzyme from Streptomyces fradiae (SFT), which is considered to be the active-site serine, was replaced with alanine by site-directed mutagenesis to improve the affinity chromatography step for a Kazal-type trypsin inhibitor pancreatic secretory trypsin inhibitor (PSTI). The resulting mutant SFT, designated as [S170A]SFT, was expressed in Streptomyces lividans and purified to homogeneity. [S170A]SFT was catalytically inactive, but still had the ability to bind tightly to PSTI and to soybean trypsin inhibitor with dissociation constants of 3.1 x 10(-7) M and 1.9 x 10(-8) M respectively. We further demonstrated that recombinant human PSTI secreted into Saccharomyces cerevisiae culture broth could be purified to homogeneity with a one-step [S170A]SFT-affinity column. The purified PSTI contained no molecules intramolecularly cleaved by active trypsin, which are found when trypsin-affinity chromatography is used for the purification. This eliminated the need for further separation of intact PSTI from intramolecularly cleaved PSTI by high-performance liquid chromatography, thus simplifying and improving its purification process.

  9. Separation and purification of epigallocatechin-3-gallate (EGCG) from green tea using combined macroporous resin and polyamide column chromatography.

    PubMed

    Jin, Xin; Liu, Mingyan; Chen, Zaixing; Mao, Ruikun; Xiao, Qinghuan; Gao, Hua; Wei, Minjie

    2015-10-01

    Epigallocatechin-3-gallate (EGCG) is a major bioactive ingredient of green tea that produces beneficial neuroprotective effects. In this paper, to optimize the EGCG enrichment, thirteen macroporous resins with different chemical and physical properties were systemically evaluated. Among the thirteen tested resins, the H-bond resin HPD826 exhibited best adsorption/desorption capabilities and desorption ratio, as well as weakest affinity for caffeine. The absorption of EGCG on the HPD826 resin followed the pseudo-second-order kinetics and Langmuir isotherm model. The separation parameters of EGCG were optimized by dynamic adsorption/desorption experiments with the HPD826 resin column. Under the optimal condition, the content of EGCG in the 30% ethanol eluent increased by 5.8-fold from 7.7% to 44.6%, with the recovery yield of 72.1%. After further purification on a polyamide column, EGCG with 74.8% purity was obtained in the 40-50% ethanol fraction with a recovery rate of 88.4%. In addition, EGCG with 95.1% purity could be easily obtained after one-step crystallization in distilled water. Our study suggests that the combined macroporous resin and polyamide column chromatography is a simple method for large-scale separation and purification of EGCG from natural plants for food and pharmaceutical applications.

  10. Separation and purification of epigallocatechin-3-gallate (EGCG) from green tea using combined macroporous resin and polyamide column chromatography.

    PubMed

    Jin, Xin; Liu, Mingyan; Chen, Zaixing; Mao, Ruikun; Xiao, Qinghuan; Gao, Hua; Wei, Minjie

    2015-10-01

    Epigallocatechin-3-gallate (EGCG) is a major bioactive ingredient of green tea that produces beneficial neuroprotective effects. In this paper, to optimize the EGCG enrichment, thirteen macroporous resins with different chemical and physical properties were systemically evaluated. Among the thirteen tested resins, the H-bond resin HPD826 exhibited best adsorption/desorption capabilities and desorption ratio, as well as weakest affinity for caffeine. The absorption of EGCG on the HPD826 resin followed the pseudo-second-order kinetics and Langmuir isotherm model. The separation parameters of EGCG were optimized by dynamic adsorption/desorption experiments with the HPD826 resin column. Under the optimal condition, the content of EGCG in the 30% ethanol eluent increased by 5.8-fold from 7.7% to 44.6%, with the recovery yield of 72.1%. After further purification on a polyamide column, EGCG with 74.8% purity was obtained in the 40-50% ethanol fraction with a recovery rate of 88.4%. In addition, EGCG with 95.1% purity could be easily obtained after one-step crystallization in distilled water. Our study suggests that the combined macroporous resin and polyamide column chromatography is a simple method for large-scale separation and purification of EGCG from natural plants for food and pharmaceutical applications. PMID:26319304

  11. Expanded bed chromatography of proteins in small-diameter columns. II. Methods development and scale up.

    PubMed

    Ghose, S; Chase, H

    2000-01-01

    The scaled down system developed in Part I of this series was further validated by using a 1-cm diameter column for method development studies for the separation of two model proteins, alcohol dehydrogenase and alpha-glucosidase, from unclarified yeast homogenate by hydrophobic interaction expanded bed chromatography based on the STREAMLINE matrix. The efficacy of solids removal and establishment of optimal binding and separation condition by stepwise elution were investigated. Equilibration of the EBA column and loading at high salt strengths affected the subsequent recovery of the two target proteins. Although good resolution between the target proteins could be achieved, peak tailing was found to be a consistent problem. The optimised separation protocol was scaled up 25-fold to a column diameter of 5.0 cm. The results were in good agreement with the run conducted in the 1-cm column, indicating the potential of using the small columns as an viable approach for method scouting and development studies.

  12. TiO2-ZrO2 affinity chromatography polymeric microchip for phosphopeptide enrichment and separation.

    PubMed

    Tsougeni, Katerina; Zerefos, Panagiotis; Tserepi, Angeliki; Vlahou, Antonia; Garbis, Spiros D; Gogolides, Evangelos

    2011-09-21

    We fabricated a TiO(2)-ZrO(2) affinity chromatography micro-column on 2 mm PMMA plates, and demonstrated the enrichment and separation of (a) a standard mono- and tetra-phosphopeptide, and (b) phosphopeptides contained in a tryptic digest of β-Casein. The chromatography column consisted of 32 parallel microchannels with common input and output ports and was fabricated by lithography directly on the polymeric substrate followed by plasma etching (i.e. standard MEMS processing) and sealed with lamination. The liquid deposited TiO(2)-ZrO(2) stationary phase was characterized by X-ray diffraction and was found to be mostly TiO(2) and ZrO(2) in crystalline phases. Off-chip UV detection and MALDI MS identification of the separated effluents were used. The chip had a capacity of >1.4 μg (0.7 nmol) of a prototype mono-phosphopeptide and a recovery of 94 ± 3%, and can be used with small samples (less than 0.1 μL depending on the syringe pump used). The chip design allows an expansion of its capacity by means of increasing the number of parallel microchannels at a constant sample volume. Our approach provided an alternative to off-line extraction tips (with typical capacities of 1-2 μg and sample volumes of 1-10 μL), and to on-chip efforts based on packed bed and frit formats. PMID:21796280

  13. Chromatography of arthropod-borne viruses on calcium phosphate columns*

    PubMed Central

    Smith, C. E. Gordon; Holt, Dolores

    1961-01-01

    This is an interim report on the fractionation of arthropod-borne viruses of groups A and B by chromatography on calcium phosphate. The method used provides an excellent, cheap and simple tool for the preparation of stable haemagglutinating and complement-fixing antigens for routine diagnostic and other purposes and for the concentration of such products. In the results reported, viruses of groups A and B have been shown to have two haemagglutinins, one of which is the virus particle. The haemagglutinins are antigenically similar but differ in sedimentation characteristics and in reaction with protamine sulfate. Group B viruses have also been shown to have two complement-fixing antigens with different sedimentation properties; one of these antigens is the virus particle. So far no complement-fixing antigen other than the virus particle has been found with group A viruses. ImagesFIG. 1 PMID:20604091

  14. Affinity chromatography using 2' fluoro-substituted RNAs for detection of RNA-protein interactions in RNase-rich or RNase-treated extracts.

    PubMed

    Hovhannisyan, Ruben; Carstens, Russ

    2009-02-01

    Use of RNA affinity chromatography is commonly used to identify RNA binding proteins that interact with specific RNA cis-elements that function in post-transcriptional gene regulation. These purifications can be complicated by residual RNase activity in cellular extracts that can degrade the RNAs on these affinity columns. Furthermore, some proteins may associate indirectly with the column as a component of multi-protein complexes that are "tethered" through the binding of cellular RNAs. We present a protocol for an RNA affinity procedure that can be used in conjunction with RNase-rich or RNase-treated extracts by using RNAs synthesized with 2' fluoro-substituted cytidine triphosphate (CTP) and uridine triphosphate (UTP). The resulting RNAs are shown to be RNase A-resistant and capable of direct coupling to adipic acid dihydrazide agarose beads. Using an RNA cis-element previously shown to bind hnRNP M, we demonstrated that the substituted RNAs preserve binding capability by a common class of RNA binding proteins. Our results provide a method that may be used more generally for RNA affinity purification or as a validation step to verify more direct binding of a given RNA binding protein to a target RNA. PMID:19317654

  15. The fabrication of all-silicon micro gas chromatography columns using gold diffusion eutectic bonding

    NASA Astrophysics Data System (ADS)

    Radadia, A. D.; Salehi-Khojin, A.; Masel, R. I.; Shannon, M. A.

    2010-01-01

    Temperature programming of gas chromatography (GC) separation columns accelerates the elution rate of chemical species through the column, increasing the speed of analysis, and hence making it a favorable technique to speedup separations in microfabricated GCs (micro-GC). Temperature-programmed separations would be preferred in an all-silicon micro-column compared to a silicon-Pyrex® micro-column given that the thermal conductivity and diffusivity of silicon is 2 orders of magnitude higher than Pyrex®. This paper demonstrates how to fabricate all-silicon micro-columns that can withstand the temperature cycling required for temperature-programmed separations. The columns were sealed using a novel bonding process where they were first bonded using a gold eutectic bond, then annealed at 1100 °C to allow gold diffusion into silicon and form what we call a gold diffusion eutectic bond. The gold diffusion eutectic-bonded micro-columns when examined using scanning electron microscopy (SEM), scanning acoustic microscopy (SAM) and blade insertion techniques showed bonding strength comparable to the previously reported anodic-bonded columns. Gas chromatography-based methane injections were also used as a novel way to investigate proper sealing between channels. A unique methane elution peak at various carrier gas inlet pressures demonstrated the suitability of gold diffusion eutectic-bonded channels as micro-GC columns. The application of gold diffusion eutectic-bonded all-silicon micro-columns to temperature-programmed separations (120 °C min-1) was demonstrated with the near-baseline separation of n-C6 to n-C12 alkanes in 35 s.

  16. Evaluation of column hardware on liquid chromatography-mass spectrometry of phosphorylated compounds.

    PubMed

    Sakamaki, Hiroshi; Uchida, Takeharu; Lim, Lee Wah; Takeuchi, Toyohide

    2015-02-13

    The influences of column hardware, such as chromatographic tubes and frits, on liquid chromatography-mass spectrometry (LC-MS) analysis of phosphorylated compounds were evaluated. The signal to noise ratio (S/N) and the intensity of flavin adenine dinucleotide (FAD) using a glass lined tube and polyethylene frit (GL-PE) column was approximately 170 and 90 times higher, respectively, than those using conventional stainless steel tube and stainless steel frit (S-S) column. In addition, the retention time of FAD using GL-PE column was the shortest compared to other columns. Interaction between phosphorylated compounds and metal ions in the flow path in the S-S column was stronger than that between them and the GL-PE column. Thus, the metal ions in the flow path in GL-PE column were low. Since the specific surface area of a pair of frits was 70 times larger than that of a chromatographic tube (150 mm×2.1 mm), the frits were found to have more effective improvement of the S/N as well as the intensity than the chromatographic tubes, when phosphorylated compounds were analyzed by LC-MS. When the evaluated phosphorylated compounds were analyzed by LC-MS(/MS) using a GL-PE column, the intensity and S/N were increased.

  17. Packed column supercritical fluid chromatography using stainless steel particles and water as a stationary phase.

    PubMed

    Murakami, Jillian N; Thurbide, Kevin B

    2015-09-15

    Stainless steel (SS) particles were demonstrated as a novel useful support for a water stationary phase in packed column supercritical fluid chromatography using a CO2 mobile phase. Separations employed flame ionization detection, and the system was operated over a range of temperatures and pressures. Retention times reproduced well with RSD values of 2.6% or less. Compared to analogous separations employing a water stationary phase coated onto a SS capillary column, the packed column method provided separations that were about 10× faster, with nearly 8-fold larger analyte retention factors, while maintaining good peak shape and comparable column efficiency. Under normal operating conditions, the packed column contains about 131 ± 4 μL/m of water phase (around a 5% m/m coating), which is over 25× greater than the capillary column and also affords it a 20-fold larger sample capacity. Several applications of the packed column system are examined, and the results indicate that it is a useful alternative to the capillary column mode, particularly where analyte loads or sample matrix interference is a concern. Given its high sample capacity, this packed column method may also be useful to explore on a more preparative scale in the future.

  18. A Computer-Interfaced Drop Counter as an Inexpensive Fraction Collector for Column Chromatography

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2008-01-01

    A computer-interfaced drop counter is described that serves as an inexpensive alternative to a fraction collector for column chromatography experiments. Undergraduate biochemistry laboratories frequently do not have the budget to purchase fraction collectors. Protocols that call for the manual measurement of fraction volumes as well as the manual…

  19. Separation of the Carotenoid Bixin from Annatto Seeds Using Thin-Layer and Column Chromatography

    ERIC Educational Resources Information Center

    McCullagh, James V.; Ramos, Nicholas

    2008-01-01

    In this experiment the carotenoid bixin is isolated from annatto ("Bixa orellana") seeds using column chromatography. The experiment has several key advantages over previous pigment separation experiments. First, unlike other experiments significant quantities of the carotenoid (typically 20 to 25 mg) can be isolated from small quantities of plant…

  20. 5-O-caffeoylshikimic acid from Solanum somalense leaves: advantage of centrifugal partition chromatography over conventional column chromatography.

    PubMed

    Chideh, Saïda; Pilard, Serge; Attoumbré, Jacques; Saguez, Robert; Hassan-Abdallah, Alshaimaa; Cailleu, Dominique; Wadouachi, Anne; Baltora-Rosset, Sylvie

    2014-09-01

    Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3/MeOH/H2O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves.

  1. 5-O-caffeoylshikimic acid from Solanum somalense leaves: advantage of centrifugal partition chromatography over conventional column chromatography.

    PubMed

    Chideh, Saïda; Pilard, Serge; Attoumbré, Jacques; Saguez, Robert; Hassan-Abdallah, Alshaimaa; Cailleu, Dominique; Wadouachi, Anne; Baltora-Rosset, Sylvie

    2014-09-01

    Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3/MeOH/H2O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves. PMID:24962011

  2. Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.

    PubMed

    Cass, Brian; Pham, Phuong Lan; Kamen, Amine; Durocher, Yves

    2005-03-01

    Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. PMID:15721774

  3. Fabry-Pérot cavity sensors for multipoint on-column micro gas chromatography detection.

    PubMed

    Liu, Jing; Sun, Yuze; Howard, Daniel J; Frye-Mason, Greg; Thompson, Aaron K; Ja, Shiou-Jyh; Wang, Siao-Kwan; Bai, Mengjun; Taub, Haskell; Almasri, Mahmoud; Fan, Xudong

    2010-06-01

    We developed and characterized a Fabry-Pérot (FP) sensor module based micro gas chromatography (microGC) detector for multipoint on-column detection. The FP sensor was fabricated by depositing a thin layer of metal and a layer of gas-sensitive polymer consecutively on the endface of an optical fiber, which formed the FP cavity. Light partially reflected from the metal layer and the polymer-air interface generated an interference spectrum, which shifted as the polymer layer absorbed the gas analyte. The FP sensor module was then assembled by inserting the FP sensor into a hole drilled in the wall of a fused-silica capillary, which can be easily connected to the conventional gas chromatography (GC) column through a universal quick seal column connector, thus enabling on-column real-time detection. We characterized the FP sensor module based microGC detector. Sensitive detection of various gas analytes was achieved with subnanogram detection limits. The rapid separation capability of the FP sensor module assembled with both single- and tandem-column systems was demonstrated, in which gas analytes having a wide range of polarities and volatilities were well-resolved. The tandem-column system obtained increased sensitivity and selectivity by employing two FP sensor modules coated with different polymers, showing great system versatility. PMID:20441156

  4. Hydrothermal preparation of hybrid carbon/silica monolithic capillary column for liquid chromatography.

    PubMed

    Yang, Peiling; Wang, Wentao; Xiao, Xing; Jia, Li

    2014-08-01

    A simple, easy and economical approach for the preparation of a hybrid carbon/silica monolithic capillary column was described for the first time by using silica monolith as framework in combination with hydrothermal carbonization at 180°C. During the preparation process, formamide was introduced to the reaction solutions to reduce the dissolution rate of monolithic silica skeleton and its optimal concentration was 1.5 M. Fourier transform infrared spectrometry, scanning electron microscopy, energy dispersive X-ray spectrometry, and inverse size exclusion chromatography were carried out to characterize the as-prepared column. The results demonstrated that carbon spheres ranging from 150 to 1000 nm were successfully attached to the surface of silica skeleton. The prepared hybrid carbon/silica column had a permeability of 4.4 × 10(-14) m(2). Chromatographic performance of the column was evaluated by separation of various compounds including alkylbenzenes, nucleosides and bases, and aromatic acids. The column exhibited an efficiency of 75,000 plates/m for butylbenzene at the optimal linear velocity of 0.23 mm/s. The successful separation of these compounds and the study on mechanism indicated that the column can be applied in mixed-mode chromatography. PMID:24830747

  5. Development and Validation of an Affinity Chromatography-Protein G Method for IgG Quantification

    PubMed Central

    Paradina Fernández, Lesly; Calvo, Loany; Viña, Lisel

    2014-01-01

    Nimotuzumab, an IgG that recognizes the epidermal growth factor receptor (EGF-R) overexpressed in some tumors, is used in the treatment of advanced head and neck cancer. For the quantification of this protein in cell culture supernatants, protein G-HPLC affinity chromatography is used due to its high affinity and specificity for antibodies of this class. The technique relies on the comparison of the area under the curve of the elution peak of the samples to be evaluated versus to a calibration curve of well-known concentrations and was validated by assessment of its robustness, specificity, repeatability, intermediate precision, accuracy, linearity, limit of detection, limit of quantification, and range. According to results of the study all validation parameters fulfilled the preestablished acceptance criteria and demonstrated the feasibility of the assay for the analysis of samples of cell culture supernatant as well as drug product. PMID:27379284

  6. Crosslinked glass fiber affinity membrane chromatography and its application to fibronectin separation.

    PubMed

    Guo, Wei; Ruckenstein, Eli

    2003-09-25

    Macroporous glass membranes were prepared from glass fiber filters via chemical crosslinking and modification, and used for the membrane affinity chromatography of fibronectin from human blood plasma. The filters were first treated with a piranha solution (a concentrated solution of H2SO4 + H2O2 in water), and then crosslinked with bifunctional organosilanes and modified to introduce amino or aniline moieties. Ligand immobilizations via diazotization and glutaraldehyde pathways were carried out and compared. Characterization of the membranes was performed using bovine serum albumin and trypsin as test ligands. By using a cartridge containing gelatin immobilized affinity membranes followed by another cartridge containing heparin immobilized membranes, fibronectin from human blood plasma could be separated.

  7. Hydrophilic-subtraction model for the characterization and comparison of hydrophilic interaction liquid chromatography columns.

    PubMed

    Wang, Jixia; Guo, Zhimou; Shen, Aijin; Yu, Long; Xiao, Yuansheng; Xue, Xingya; Zhang, Xiuli; Liang, Xinmiao

    2015-06-12

    Nowadays more and more hydrophilic interaction liquid chromatography (HILIC) columns with diverse functional groups have become commercially available, which pose a challenge to select an appropriate one. However, there is no universal model to provide guidance for selecting HILIC columns. To handle this problem, a retention model named "hydrophilic-subtraction model" was developed to characterize and compare HILIC columns. The hydrophilic-subtraction model, which was designed based on the widely recognized HILIC retention mechanisms including hydrophilic partitioning, hydrogen-bonding and electrostatic interactions, was established by the retention of 41 solutes with various properties on 8 representative HILIC columns. High correlation coefficients (R(2)≥0.990) and small standard deviations (SD≤0.041) indicated that this model correlated effectively the retention with solute descriptors and column parameters. To evaluate reliability of the model, the model was further applied to characterize 15 additional HILIC columns using 41 solutes. The results of multiple linear regression confirmed the significance of the model. The regression coefficients of the model were used to investigate retention mechanisms occurring in different chromatographic systems. Based on these regression coefficients, selectivities of HILIC stationary phases were exhibited intuitively by an angle graph and a spider diagram, which could be used as guidance for researchers to select appropriate columns for HILIC separation. Additionally, a rapid and convenient procedure was proposed for characterizing HILIC columns. PMID:25935798

  8. Isolation and partial characterization of Bromelia hemisphaerica protease by affinity chromatography.

    PubMed

    Ochoa, N; Agundis, C; Córdoba, F

    1987-01-01

    Hemisphaericin, the protease from Bromelia hemisphaerica fruit juice was isolated by affinity chromatography in one step, using a mercurial sepharose derivative. The enzyme behaves as a single component in immunodifussion, immunoelectrophoresis and polyacrylamide electrophoresis in the presence of SDS and 2-mercaptoethanol. Association and dissociation of active components were evidenced in electrophoresis at pH 3.6 and at pH 8.6. Immunoelectrophoresis analyses also disclosed a certain degree of internal immunological heterogeneity. The results are explained by the presence of an enzyme subunit, of about 8000 daltons, endowed with polymeric properties induced by the pH and oxidative environment.

  9. Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography

    SciTech Connect

    Boyer, R.F.; Allen, T.L.; Dykema, P.A.

    1987-02-05

    Eight chemically modified cellulose supports were tested for their ability to adsorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography. 22 references.

  10. Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli.

    PubMed

    Velkov, Tony; Jones, Alun; Lim, Maria L R

    2008-01-01

    A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor. PMID:18800304

  11. Development of a novel affinity chromatography resin for platform purification of lambda fabs.

    PubMed

    Eifler, Nora; Medaglia, Giovanni; Anderka, Oliver; Laurin, Linus; Hermans, Pim

    2014-01-01

    Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established. PMID:25082738

  12. Demonstration of motionless Knudsen pump based micro-gas chromatography featuring micro-fabricated columns and on-column detectors.

    PubMed

    Liu, Jing; Gupta, Naveen K; Wise, Kensall D; Gianchandani, Yogesh B; Fan, Xudong

    2011-10-21

    This paper reports the investigation of a micro-gas chromatography (μGC) system that utilizes an array of miniaturized motionless Knudsen pumps (KPs) as well as microfabricated separation columns and optical detectors. A prototype system was built to achieve a flow rate of 1 mL min(-1) and 0.26 mL min(-1) for helium and dry air, respectively, when they were used as carrier gas. This system was then employed to evaluate GC performance compromises and demonstrate the ability to separate and detect gas mixtures containing analytes of different volatilities and polarities. Furthermore, the use of pressure programming of the KP array was demonstrated to significantly shorten the analysis time while maintaining a high detection resolution. Using this method, we obtained a high resolution detection of 5 alkanes of different volatilities within 5 min. Finally, we successfully detected gas mixtures of various polarities using a tandem-column μGC configuration by installing two on-column optical detectors to obtain complementary chromatograms.

  13. 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.

  14. 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.

  15. Affinity chromatography of yeast alpha-glucosidase using ligand-mediated chromatography on immobilized phenylboronic acids.

    PubMed Central

    Myöhänen, T A; Bouriotis, V; Dean, P D

    1981-01-01

    The synthesis of 3-nitro-4-(6-aminohexylamido)phenylboronic acid is described. The properties of two novel forms of immobilized phenylboronate agarose adsorbents [m-aminophenylboronic acid-Matrex Gel and 3-nitro-4-(6-aminohexylamido)phenylboronic acid-Sepharose CL-6B] were investigated. Both gels bind and selectively retard the glycoprotein alpha-glucosidase from yeast. The retardation is affected by following parameters: (i) pH, (ii) presence of sugar, (iii) concentration of sugar and (iv) buffer species (especially triethanolamine). Five sugars were studied, namely sorbitol, fructose, ribose, glucose and maltose. The concentration of sugar required to produce significant retardation increased in the above order, whereas the ability of sugar to form a complex with boron decreases in the same order. These effects were observed with crude as well as pure enzyme. Since alpha-glucosidase is a glycoprotein, it is proposed that this protein is mainly bound to these immobilized phenylboronates via sugar (glyco) residues. Displacement of the enzyme from the column is effected by the sugar in the buffer (or in a preincubation mixture). However, the marked pH-dependence (this retardation effect could only be observed at pH 7.4) suggests that these results are not due solely to hydrophobic or ionic mechanisms and are more complex than simple sugar-phenylboronic acid interactions. PMID:7034722

  16. Stereoselective Binding of Chiral Ligands to Single Nucleotide Polymorphs (SNPs) of the Human Organic Cation Transporter-1 Determined Using Cellular Membrane Affinity Chromatography

    PubMed Central

    Moaddel, R.; Bighi, F.; Yamaguchi, R.; Patel, S.; Ravichandran, S.; Wainer, I.W.

    2010-01-01

    Membranes from stably transfected cell lines that expresses two point mutations of the human organic cation 1 transporter (hOCT1), R488M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the Cellular Membrane Affinity Chromatography (CMAC) (hOCT1G465R) and CMAC(hOCT1R488M). Columns were created using both stationary phases and frontal displacement chromatography experiments were conducted using [3H]-methyl phenyl pyridinium, [3H]-MPP+, as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol and isoproterenol. The chromatographic data obtained was used to refine a previously developed pharmacophore for the hOCT1 transporter. PMID:20206116

  17. Preparation and characterization of alkyl methacrylate-based monolithic columns for capillary gas chromatography applications.

    PubMed

    Yusuf, Kareem; Aqel, Ahmad; A L Othman, Zeid; Badjah-Hadj-Ahmed, Ahmed Yacine

    2013-08-01

    Gas chromatography (GC) is considered the least common application of both polymer and silica-based monolithic columns. This study describes the fabrication of alkyl methacrylate monolithic materials for use as stationary phases in capillary gas chromatography. Following the deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (TMSM), the monoliths were formed by the co-polymerization of either hexyl methacrylate (HMA) or lauryl methacrylate (LMA) with different percentage of ethylene glycol dimethacrylate (EDMA) in presence of an initiator (azobisisobutyronitrile, AIBN) and a mixture of porogens include 1-propanol, 1,4-butanediol and water. The monoliths were prepared in 500mm length capillaries possessing inner diameters of 250μm. The efficiencies of the monolithic columns for low molecular weight compounds significantly improved as the percentage of crosslinker was increased, because of the greater proportion of pores less than 50nm. The columns containing lower percentages of crosslinker were able to rapidly separate a series of 8 alkane members in 0.7min, but the separation was less efficient for the light alkanes. Columns prepared with the lauryl methacrylate monomer yielded a different morphology for the monolith-interconnected channels. The channels were more branched, which increased the separation time, and unlike the other columns, allowed for temperature programming.

  18. Characterization of Murine Brain Membrane Glycoproteins by Detergent Assisted Lectin Affinity Chromatography (DALAC)

    PubMed Central

    Wei, Xin; Dulberger, Charles; Li, Lingjun

    2010-01-01

    Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization has been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that under certain concentrations, detergents can minimize the nonspecific bindings and facilitate the elution of hydrophobic glycoproteins. With the Detergent Assisted Lectin Affinity Chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. 699 proteins were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome. PMID:20700909

  19. p53-Encoding pDNA Purification by Affinity Chromatography for Cancer Therapy.

    PubMed

    Sousa, Ângela; Queiroz, João A; Sousa, Fani

    2015-01-01

    The gene therapy approach based on reestablishment of p53 tumor suppressor, which acts as a prevailing guardian against malignant cell transformation, is raising new prospects on the outcome of an effective anticancer treatment. It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Therefore, several downstream methods have been proposed to achieve high quantities of supercoiled plasmid DNA with pharmaceutical grade purity. Affinity chromatography with amino acids as ligands has recently yielded interesting results because these ligands take advantage of their biological function or chemical structure to promote specific interactions with different nucleic acids. Here, we describe detailed procedures for the preparation and purification of supercoiled plasmid DNA, with the purity degree required by regulatory agencies, by using arginine affinity chromatography. With this methodology pure pDNA is obtained, efficient on eukaryotic cell transfection and biologically active, resulting in the reestablishment of the p53 protein levels in cancer cell lines. PMID:26072404

  20. Column chromatography as a useful step in purification of diatom pigments.

    PubMed

    Tokarek, Wiktor; Listwan, Stanisław; Pagacz, Joanna; Leśniak, Piotr; Latowski, Dariusz

    2016-01-01

    Fucoxanthin, diadinoxanthin and diatoxanthin are carotenoids found in brown algae and most other heterokonts. These pigments are involved in photosynthetic and photoprotective reactions, and they have many potential health benefits. They can be extracted from diatom Phaeodactylum tricornutum by sonication, extraction with chloroform : methanol and preparative thin layer chromatography. We assessed the utility of an additional column chromatography step in purification of these pigments. This novel addition to the isolation protocol increased the purity of fucoxanthin and allowed for concentration of diadinoxanthin and diatoxanthin before HPLC separation. The enhanced protocol is useful for obtaining high purity pigments for biochemical studies. PMID:27486920

  1. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme. PMID:26644295

  2. Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Bering, C. Larry; Kuhns, Jennifer J.; Rowlett, Roger

    1998-08-01

    We have developed a rapid and inexpensive experiment utilizing affinity chromatography to isolate carbonic anhydrase (CA) from bovine blood. The more specific an affinity gel is the better the purification, but the greater the cost. Some costs would be prohibitive in the undergraduate biochemistry laboratory. Less specific resins may be more affordable but may bind a number of closely related proteins. One alternative would be to couple a specific ligand to an inexpensive resin such as an ion exchanger. We describe a simple procedure for preparing a sulfonamide-coupled resin which specifically binds CA from a blood hemolysate. The CA is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that only a single band of 31 kD was obtained. The instructor can readily prepare the affinity gel prior to the lab, and the students, beginning with packed red blood cells can carry out the lysis, binding to the gel, elution, enzymatic assays, and electrophoresis.

  3. Weak affinity chromatography as a new approach for fragment screening in drug discovery.

    PubMed

    Duong-Thi, Minh-Dao; Meiby, Elinor; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2011-07-01

    Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM-μM in dissociation constant (K(D))) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM-10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC-MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening. PMID:21352794

  4. Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

    PubMed Central

    Marszałł, Michał P.; Moaddel, Ruin; Jozwiak, Krzysztof; Bernier, Michel; Wainer, Irving W.

    2008-01-01

    Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N-terminus to create the Hsp90α(NT)-column or C-terminus to create the Hsp90α(CT)-column. Binding to the exposed C-terminus on the Hsp90α(NT)-column was characterized using frontal chromatography and C-terminus ligands coumermycin A1(CA1) and novobiocin (NOVO). The calculated Kd values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Non-linear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90α complex, 22.2 (±8.8) μM−1 sec−1 and 2.7 (±0.6) sec−1, respectively. Binding to the exposed N-terminus on the Hsp90α(CT)-column was characterized using frontal chromatography. The Kd values of N-terminus ligands geldanamycin (GM) (90 ± 50 nM) 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) (210 ± 50 nM) and radicicol (RAD) (20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC50 values for inhibition of ATPase activity on the Hsp90α(CT)-column. The IC50 for GM was 2.80 ± 0.18 μM and the relative IC50 values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition. PMID:18047824

  5. 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.

  6. 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.

  7. Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

    PubMed Central

    Brehm, R D; Tranter, H S; Hambleton, P; Melling, J

    1990-01-01

    A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production. Images PMID:2339869

  8. Wide Range of Biotin (Vitamin H) Content in Foodstuffs and Powdered Milks as Assessed by High-performance Affinity Chromatography

    PubMed Central

    Hayakawa, Kou; Katsumata, Noriyuki; Abe, Kiyomi; Hirano, Masahiko; Yoshikawa, Kazuyuki; Ogata, Tsutomu; Horikawa, Reiko; Nagamine, Takeaki

    2009-01-01

    The biotin (vitamin H) contents of various foodstuffs were determined by using a newly developed high-performance affinity chromatography with a trypsin-treated avidin-bound column. Biotin was derivatized with 9-anthryldiazomethane (ADAM) to fluorescent biotin-ADAM ester. A wide range of biotin contents were found in various foodstuffs depending upon the species (strain), season, organ (of plants and animals), geography, freshness, preparation method and storage method. Among the foodstuffs and fermented foods tested, it was found that wide distributions of biotin content were observed in powdered milk, natto, sake (rice wine), beer, edible oil and sea weed. Since powdered milk is important for child health and development, 14 kinds of powdered and special milks for use in children’s diseases were intensively measured. We found that several special milk powders for children with allergies contained low levels of free biotin. Use of these powdered milks caused skin diseases and alopecia in some patients possessing thermolabile serum biotinidase, and administration of free biotin improved their symptoms dramatically. Therefore, it is essential to estimate the total and free biotin contents on each foodstuff in order to improve effective biotin intake and support better health and quality of life for people. PMID:24790379

  9. Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.

    PubMed

    Efremenko, E; Votchitseva, Y; Plieva, F; Galaev, I; Mattiasson, B

    2006-05-01

    Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.

  10. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

    PubMed

    Smits, Nathalie G E; Blokland, Marco H; Wubs, Klaas L; Nessen, Merel A; van Ginkel, Leen A; Nielen, Michel W F

    2015-08-01

    The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST. PMID:26077745

  11. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

    SciTech Connect

    Niture, Suryakant K.; Doneanu, Catalin E.; Velu, Chinavenmeni S.; Bailey, Nathan I.; Srivenugopal, Kalkunte S. . E-mail: Kalkunte.srivenugopal@ttuhsc.edu

    2005-12-02

    Recent evidence suggests that human O {sup 6}-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase {delta}, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} and {beta}, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

  12. Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.

    PubMed

    Efremenko, E; Votchitseva, Y; Plieva, F; Galaev, I; Mattiasson, B

    2006-05-01

    Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration. PMID:16088350

  13. High performance mini-gas chromatography-flame ionization detector system based on micro gas chromatography column.

    PubMed

    Zhu, Xiaofeng; Sun, Jianhai; Ning, Zhanwu; Zhang, Yanni; Liu, Jinhua

    2016-04-01

    Monitoring Volatile organic compounds (VOCs) was a very important measure for preventing environmental pollution, therefore, a mini gas chromatography (GC) flame ionization detector (FID) system integrated with a mini H2 generator and a micro GC column was developed for environmental VOC monitoring. In addition, the mini H2 generator was able to make the system explode from far away due to the abandoned use of a high pressure H2 source. The experimental result indicates that the fabricated mini GC FID system demonstrated high repeatability and very good linear response, and was able to rapidly monitor complicated environmental VOC samples.

  14. High performance mini-gas chromatography-flame ionization detector system based on micro gas chromatography column

    NASA Astrophysics Data System (ADS)

    Zhu, Xiaofeng; Sun, Jianhai; Ning, Zhanwu; Zhang, Yanni; Liu, Jinhua

    2016-04-01

    Monitoring Volatile organic compounds (VOCs) was a very important measure for preventing environmental pollution, therefore, a mini gas chromatography (GC) flame ionization detector (FID) system integrated with a mini H2 generator and a micro GC column was developed for environmental VOC monitoring. In addition, the mini H2 generator was able to make the system explode from far away due to the abandoned use of a high pressure H2 source. The experimental result indicates that the fabricated mini GC FID system demonstrated high repeatability and very good linear response, and was able to rapidly monitor complicated environmental VOC samples.

  15. Amine Gradient Stationary Phases on In-House Built Monolithic Columns for Liquid Chromatography.

    PubMed

    Dewoolkar, Veeren C; Jeong, Lena N; Cook, Daniel W; Ashraf, Kayesh M; Rutan, Sarah C; Collinson, Maryanne M

    2016-06-01

    Stationary phase gradients on monolithic silica columns have been successfully and reproducibly prepared and characterized with comparisons made to uniformly modified stationary phases. Stationary phase gradients hold great potential for use in liquid chromatography (LC), both in terms of simplifying analysis as well as providing novel selectivity. In this work, we demonstrate the creation of a continuous stationary phase gradient on in-house synthesized monolithic columns by infusing an aminoalkoxysilane solution through the silica monoliths via controlled rate infusion. The presence of amine and its distribution along the length of gradient and uniformly modified columns were assessed via X-ray photoelectron spectroscopy (XPS). XPS showed a clear gradient in surface coverage along the length of the column for the gradient stationary phases while a near uniform distribution on the uniformly modified stationary phases. To demonstrate the application of these gradient stationary phases, the separations of both nucleobases and weak acids/weak bases on these gradient stationary phases have been compared to uniformly modified and unmodified silica columns. Of particular note, the retention characteristics of 11 gradient columns, 5 uniformly modified columns, and 5 unmodified columns have been tested to establish the reproducibility of the synthetic procedures. Standard deviations of the retention factors were in the range from 0.06 to 0.5, depending on the analyte species. We show that selectivity is achieved with the stationary phase gradients that are significantly different from either uniformly modified amine or unmodified columns. These results indicate the significant promise of this strategy for creating novel stationary phases for LC. PMID:27203513

  16. Signal analysis of NEMS sensors at the output of a chromatography column

    SciTech Connect

    Bertholon, François; Harant, Olivier; Bourlon, Bertrand; Gerfault, Laurent; Grangeat, Pierre; Jutten, Christian

    2015-01-13

    This article introduces a joined Bayesian estimation of gas samples issued from a gas chromatography column (GC) coupled with a NEMS sensor based on Giddings Eyring microscopic molecular stochastic model. The posterior distribution is sampled using a Monte Carlo Markov Chain and Gibbs sampling. Parameters are estimated using the posterior mean. This estimation scheme is finally applied on simulated and real datasets using this molecular stochastic forward model.

  17. Signal analysis of NEMS sensors at the output of a chromatography column

    NASA Astrophysics Data System (ADS)

    Bertholon, François; Harant, Olivier; Jutten, Christian; Bourlon, Bertrand; Gerfault, Laurent; Grangeat, Pierre

    2015-01-01

    This article introduces a joined Bayesian estimation of gas samples issued from a gas chromatography column (GC) coupled with a NEMS sensor based on Giddings Eyring microscopic molecular stochastic model. The posterior distribution is sampled using a Monte Carlo Markov Chain and Gibbs sampling. Parameters are estimated using the posterior mean. This estimation scheme is finally applied on simulated and real datasets using this molecular stochastic forward model.

  18. Flow rate dependent extra-column variance from injection in capillary liquid chromatography.

    PubMed

    Aggarwal, Pankaj; Liu, Kun; Sharma, Sonika; Lawson, John S; Dennis Tolley, H; Lee, Milton L

    2015-02-01

    Efficiency and resolution in capillary liquid chromatography (LC) can be significantly affected by extra-column band broadening, especially for isocratic separations. This is particularly a concern in evaluating column bed structure using non-retained test compounds. The band broadening due to an injector supplied with a commercially available capillary LC system was characterized from experimental measurements. The extra-column variance from the injection valve was found to have an extra-column contribution independent of the injection volume, showing an exponential dependence on flow rate. The overall extra-column variance from the injection valve was found to vary from 34 to 23 nL. A new mathematical model was derived that explains this exponential contribution of extra-column variance on chromatographic performance. The chromatographic efficiency was compromised by ∼130% for a non-retained analyte because of injection valve dead volume. The measured chromatographic efficiency was greatly improved when a new nano-flow pumping system with integrated injection valve was used.

  19. Core-Shell Columns in High-Performance Liquid Chromatography: Food Analysis Applications

    PubMed Central

    Preti, Raffaella

    2016-01-01

    The increased separation efficiency provided by the new technology of column packed with core-shell particles in high-performance liquid chromatography (HPLC) has resulted in their widespread diffusion in several analytical fields: from pharmaceutical, biological, environmental, and toxicological. The present paper presents their most recent applications in food analysis. Their use has proved to be particularly advantageous for the determination of compounds at trace levels or when a large amount of samples must be analyzed fast using reliable and solvent-saving apparatus. The literature hereby described shows how the outstanding performances provided by core-shell particles column on a traditional HPLC instruments are comparable to those obtained with a costly UHPLC instrumentation, making this novel column a promising key tool in food analysis. PMID:27143972

  20. Gas chromatography on wall-coated open-tubular columns with ionic liquid stationary phases.

    PubMed

    Poole, Colin F; Lenca, Nicole

    2014-08-29

    Ionic liquids have moved from novel to practical stationary phases for gas chromatography with an increasing portfolio of applications. Ionic liquids complement conventional stationary phases because of a combination of thermophysical and solvation properties that only exist for ionic solvents. Their high thermal stability and low vapor pressure makes them suitable as polar stationary phases for separations requiring high temperatures. Ionic liquids are good solvents and can be used to expand the chemical space for separations. They are the only stationary phases with significant hydrogen-bond acidity in common use; they extend the hydrogen-bond basicity of conventional stationary phases; they are as dipolar/polarizable as the most polar conventional stationary phases; and some ionic liquids are significantly less cohesive than conventional polar stationary phases. Problems in column coating techniques and related low column performance, column activity, and stationary phase reactivity require further exploration as the reasons for these features are poorly understood at present.

  1. Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography ▿ † ‡

    PubMed Central

    Robichon, Carine; Luo, Jianying; Causey, Thomas B.; Benner, Jack S.; Samuelson, James C.

    2011-01-01

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein. PMID:21602383

  2. Novel design for centrifugal counter-current chromatography: VI. Ellipsoid column

    PubMed Central

    Gu, Dongyu; Yang, Yi; Xin, Xuelei; Aisa, Haji Akber; Ito, Yoichiro

    2014-01-01

    A novel ellipsoid column was designed for centrifugal counter-current chromatography. Performance of the ellipsoid column with a capacity of 3.4 mL was examined with three different solvent systems composed of 1-butanol-acetic acid-water (4:1:5, v/v) (BAW), hexane-ethyl acetate-methanol-0.1 M HCl (1:1:1:1, v/v) (HEMH), and 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate in water (PEG-DPP) each with suitable test samples. In dipeptide separation with BAW system, both stationary phase retention (Sf) and peak resolution (Rs) of the ellipsoid column were much higher at 0° column angle (column axis parallel to the centrifugal force) than at 90° column angle (column axis perpendicular to the centrifugal force), where elution with the lower phase at a low flow rate produced the best separation yielding Rs at 2.02 with 27.8% Sf at a flow rate of 0.07 ml/min. In the DNP-amino acid separation with HEMW system, the best results were obtained at a flow rate of 0.05 ml/min with 31.6% Sf yielding high Rs values at 2.16 between DNP-DL-glu and DNP-β-ala peaks and 1.81 between DNP-β-ala and DNP-L-ala peaks. In protein separation with PEG-DPP system, lysozyme and myolobin were resolved at Rs of 1.08 at a flow rate of 0.03 ml/min with 38.9% Sf. Most of those Rs values exceed those obtained from the figure-8 column under similar experimental conditions previously reported. PMID:25309116

  3. Pressure Tuning of First Dimension Columns in Comprehensive Two-Dimensional Gas Chromatography.

    PubMed

    Sharif, Khan M; Kulsing, Chadin; Marriott, Philip J

    2016-09-20

    The experimental approach and mechanism of pressure tuning (PT) are introduced for the first stage of a comprehensive two-dimensional gas chromatography (GC × GC) separation. The PT-GC × GC system incorporates a first dimension ((1)D) coupled column ensemble comprising a pair of (1)D columns ((1)D1 and (1)D2) connected via a microfluidic splitter device, allowing variable decompression of carrier gas across each (1)D column, and a conventional (2)D narrow bore column. By variation of junction pressure between the (1)D1 and (1)D2 columns, tunable total (1)D retentions of analytes are readily derived. Separations of a standard mixture comprising a number of different chemical classes (including alkanes, monoaromatics, alcohols, aldehydes, ketones, and esters) and Australian tea tree oil (TTO) were studied as practical examples of the PT-GC × GC system application. This illustrated the change of analyte retention time with experimental conditions depending on void time and retention on the different columns. In addition to void time change, variation of carrier gas relative decompression in the (1)D ensemble leads to tunable contribution of the (1)D1/(1)D2 columns that changes apparent polarity and selectivity of the ensemble. The resulting changes in (1)D elution order further altered elution temperature and thus retention of each analyte on the (2)D column in temperature-programmed GC × GC. 2D orthogonality measurements were then conducted to evaluate overall separation performance under application of different (1)D junction pressure. As a result, distribution and selectivity of particular target compounds, monoterpenes, sesquiterpenes, and oxygenated terpenes in 2D space, and thus orthogonality, could be adequately tuned. This indicates the potential of PT-GC × GC to be applicable for practical sample separation and provides a general approach to tune selectivity of target compounds.

  4. Pressure Tuning of First Dimension Columns in Comprehensive Two-Dimensional Gas Chromatography.

    PubMed

    Sharif, Khan M; Kulsing, Chadin; Marriott, Philip J

    2016-09-20

    The experimental approach and mechanism of pressure tuning (PT) are introduced for the first stage of a comprehensive two-dimensional gas chromatography (GC × GC) separation. The PT-GC × GC system incorporates a first dimension ((1)D) coupled column ensemble comprising a pair of (1)D columns ((1)D1 and (1)D2) connected via a microfluidic splitter device, allowing variable decompression of carrier gas across each (1)D column, and a conventional (2)D narrow bore column. By variation of junction pressure between the (1)D1 and (1)D2 columns, tunable total (1)D retentions of analytes are readily derived. Separations of a standard mixture comprising a number of different chemical classes (including alkanes, monoaromatics, alcohols, aldehydes, ketones, and esters) and Australian tea tree oil (TTO) were studied as practical examples of the PT-GC × GC system application. This illustrated the change of analyte retention time with experimental conditions depending on void time and retention on the different columns. In addition to void time change, variation of carrier gas relative decompression in the (1)D ensemble leads to tunable contribution of the (1)D1/(1)D2 columns that changes apparent polarity and selectivity of the ensemble. The resulting changes in (1)D elution order further altered elution temperature and thus retention of each analyte on the (2)D column in temperature-programmed GC × GC. 2D orthogonality measurements were then conducted to evaluate overall separation performance under application of different (1)D junction pressure. As a result, distribution and selectivity of particular target compounds, monoterpenes, sesquiterpenes, and oxygenated terpenes in 2D space, and thus orthogonality, could be adequately tuned. This indicates the potential of PT-GC × GC to be applicable for practical sample separation and provides a general approach to tune selectivity of target compounds. PMID:27548569

  5. Novel design for centrifugal counter-current chromatography: VI. Ellipsoid column.

    PubMed

    Gu, Dongyu; Yang, Yi; Xin, Xuelei; Aisa, Haji Akber; Ito, Yoichiro

    2015-01-01

    A novel ellipsoid column was designed for centrifugal counter-current chromatography. Performance of the ellipsoid column with a capacity of 3.4 mL was examined with three different solvent systems composed of 1-butanol-acetic acid-water (4:1:5, v/v) (BAW), hexane-ethyl acetate-methanol-0.1 M HCl (1:1:1:1, v/v) (HEMH), and 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate in water (PEG-DPP) each with suitable test samples. In dipeptide separation with BAW system, both stationary phase retention (Sf) and peak resolution (Rs) of the ellipsoid column were much higher at 0° column angle (column axis parallel to the centrifugal force) than at 90° column angle (column axis perpendicular to the centrifugal force), where elution with the lower phase at a low flow rate produced the best separation yielding Rs at 2.02 with 27.8% Sf at a flow rate of 0.07 ml/min. In the DNP-amino acid separation with HEMW system, the best results were obtained at a flow rate of 0.05 ml/min with 31.6% Sf yielding high Rs values at 2.16 between DNP-DL-glu and DNP-β-ala peaks and 1.81 between DNP-β-ala and DNP-L-ala peaks. In protein separation with PEG-DPP system, lysozyme and myolobin were resolved at Rs of 1.08 at a flow rate of 0.03 ml/min with 38.9% Sf. Most of those Rs values exceed those obtained from the figure-8 column under similar experimental conditions previously reported.

  6. ENANTIOMER SEPARATION OF POLYCHLORINATED BIPHENYL ATROPISOMERS AND POLYCHLORINATED BIPHENYL RETENTION BEHAVIOR ON MODIFIED CYCLODEXTRIN CAPILLARY GAS CHROMATOGRAPHY COLUMNS

    EPA Science Inventory

    Seven commercially-available chiral capillary gas chromatography columns containing modified cyclodextrins were evaluated for their ability to separate enantiomers of the 19 stable chiral polychlorinated biphenyl (PCB) atropisomers, and for their ability to separate these enantio...

  7. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  8. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    PubMed

    Rimmelzwaan, G F; Groen, J; Juntti, N; Teppema, J S; UytdeHaag, F G; Osterhaus, A D

    1987-03-01

    Immuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infectivity titration system, in an ELISA, in a haemagglutination assay and by negative contrast electron microscopy to quantify CPV or CPV antigen. The degree of purification was further estimated by testing the fractions for total protein content in a colorimetric method, for bovine serum albumin content in an ELISA and by SDS-PAGE. Over 99% of the contaminating proteins proved to be removed, and 20% or 70-90% of infectious CPV or CPV antigen, respectively, was recovered.

  9. HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND THE ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS: BINDING OF GLICLAZIDE WITH GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high affinity sites (average Ka, 7.1-10 × 104 M−1) and a group of lower affinity sites (average Ka, 5.7-8.9 × 103 M−1) at pH 7.4 and 37°C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the Ka values for gliclazide at these sites being 1.9 × 104 M−1 and 6.0 × 104 M−1, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification. PMID:21922305

  10. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  11. Purification of peroxidase from red cabbage (Brassica oleracea var. capitata f. rubra) by affinity chromatography.

    PubMed

    Somtürk, Burcu; Kalın, Ramazan; Özdemir, Nalan

    2014-08-01

    Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9% from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K m and V max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC50 and K i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702±0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.

  12. High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

    PubMed

    Li, Zhao; Beeram, Sandya R; Bi, Cong; Suresh, D; Zheng, Xiwei; Hage, David S

    2016-01-01

    The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples. PMID:26827600

  13. 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

  14. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    PubMed

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-01

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. PMID:25981289

  15. DETECTION OF HETEROGENEOUS DRUG-PROTEIN BINDING BY FRONTAL ANALYSIS AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Tong, Zenghan; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous binding in biomolecular interactions, using the binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of binding heterogeneity. The methods developed in this work for the detection of binding heterogeneity are not limited to drug interactions with HSA but could be applied to other types of drug-protein binding or to additional biological systems with heterogeneous binding. PMID:21612784

  16. Glycan-specific whole cell affinity chromatography: A versatile microbial adhesion platform

    PubMed Central

    Van Tassell, Maxwell L.; Price, Neil P.J.; Miller, Michael J.

    2014-01-01

    We have sought a universal platform for elucidating and exploiting specificity of glycan-mediated adhesion by potentially uncharacterized microorganisms. Several techniques exist to explore microbial interactions with carbohydrate structures. Many are unsuitable for investigating specific mechanisms or uncharacterized organisms, requiring pure cultures, labeling techniques, expensive equipment, or other limitations such as questionable stability, stereospecificity, or scalability. We have adapted an affinity chromatography resin as a model to overcome these drawbacks, among others. It readily allows for the quantification, selection, and manipulation of target organisms based on interactions with glycan ligands. To maximize its utility as a selective screening method, we have constructed the tool such that it:•Promotes whole-cell interactions using viable, unaltered cells.•Provides robust spatial interactions with target glycans, presented with controlled stereo-specificity, for high affinity/avidity interactions that reflect a complex in vivo matrix.•Has the ability to utilize any reducing glycan, is quick, efficient, safe, and affordable to construct, and is scalable and reusable for multiple applications. PMID:26150959

  17. Necator americanus secretory acetylcholinesterase and its purification from excretory-secretory products by affinity chromatography.

    PubMed

    Pritchard, D I; Leggett, K V; Rogan, M T; McKean, P G; Brown, A

    1991-03-01

    Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthiocholine iodide (ATC), and its activity was inhibited by eserine (1.1 x 10(-8) M). Ancylostoma ceylanicum, another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophageal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenates and earlier developmental stages. On IEF, purified AChE resolved predominantly with a pl of 3.55; proteins with a similar pl were recognized by rabbit anti-AChE. IgG preparations of this antiserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host-parasite relationship. PMID:2052405

  18. A versatile noninvasive method for adsorber quantification in batch and column chromatography based on the ionic capacity.

    PubMed

    Huuk, Thiemo C; Briskot, Till; Hahn, Tobias; Hubbuch, Jürgen

    2016-05-01

    Within the Quality by Design (QbD) framework proposed by the International Conference on Harmonisation (ICH), high-throughput process development (HTPD) and mechanistic modeling are of outstanding importance for future biopharmaceutical chromatography process development. In order to compare the data derived from different column scales or batch chromatographies, the amount of adsorber has to be quantified with the same noninvasive method. Similarly, an important requirement for the implementation of mechanistic modeling is the reliable determination of column characteristics such as the ionic capacity Λ for ion-exchange chromatography with the same method at all scales and formats. We developed a method to determine the ionic capacity in column and batch chromatography, based on the adsorption/desorption of the natural, uv-detectable amino acid histidine. In column chromatography, this method produces results comparable to those of classical acid-base titration. In contrast to acid-base titration, this method can be adapted to robotic batch chromatographic experiments. We are able to convert the adsorber volumes in batch chromatography to the equivalent volume of a compressed column. In a case study, we demonstrate that this method increases the quality of SMA parameters fitted to batch adsorption isotherms, and the capability to predict column breakthrough experiments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:666-677, 2016.

  19. A versatile noninvasive method for adsorber quantification in batch and column chromatography based on the ionic capacity.

    PubMed

    Huuk, Thiemo C; Briskot, Till; Hahn, Tobias; Hubbuch, Jürgen

    2016-05-01

    Within the Quality by Design (QbD) framework proposed by the International Conference on Harmonisation (ICH), high-throughput process development (HTPD) and mechanistic modeling are of outstanding importance for future biopharmaceutical chromatography process development. In order to compare the data derived from different column scales or batch chromatographies, the amount of adsorber has to be quantified with the same noninvasive method. Similarly, an important requirement for the implementation of mechanistic modeling is the reliable determination of column characteristics such as the ionic capacity Λ for ion-exchange chromatography with the same method at all scales and formats. We developed a method to determine the ionic capacity in column and batch chromatography, based on the adsorption/desorption of the natural, uv-detectable amino acid histidine. In column chromatography, this method produces results comparable to those of classical acid-base titration. In contrast to acid-base titration, this method can be adapted to robotic batch chromatographic experiments. We are able to convert the adsorber volumes in batch chromatography to the equivalent volume of a compressed column. In a case study, we demonstrate that this method increases the quality of SMA parameters fitted to batch adsorption isotherms, and the capability to predict column breakthrough experiments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:666-677, 2016. PMID:27324662

  20. Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

    PubMed Central

    Jackson, Abby J.; Anguizola, Jeanethe; Pfaunmiller, Erika L.; Hage, David S.

    2013-01-01

    Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions. PMID:23657448

  1. High resolution capillary column development for selective separations in gas chromatography

    SciTech Connect

    Przybyciel, M.

    1985-01-01

    A review of techniques for the preparation of high resolution capillary columns for gas chromatography is presented. Surface roughing, surface deactivation, stationary phase coating, and stationary phase crosslinking are discussed. Criteria for the selection of GC stationary phases and procedures for column evaluation are presented. A method is proposed for the isolation and determination of crude oil contamination in tropical plants and sediments. The method uses Florisil (TM) chromatography for the simultaneous clean-up and fractionation of aliphatic and aromatic hydrocarbons. Crosslinked SE-54 fused silica capillary columns prepared in our laboratory were employed for all GC separations. Mass spectrometry was used to help locate and identify specific oil components despite the intense background of the chromatogram. Crude oil components were identified in extracts of mangrove plant samples collected from the Peck Slip oil spill site at Media Munda, Puerto Rico. Crude oil components were also identified in sediment samples from controlled oil spill of Prudhoe Bay oil at Laguna de Chiriqui, Panama.

  2. Column chromatography of human small-intestinal maltase, isomaltase and invertase activities

    PubMed Central

    Dahlqvist, A.; Telenius, U.

    1969-01-01

    1. The maltase, isomaltase and invertase (sucrase) activities of solubilized mucosal preparations from human jejunum and ileum were studied with column chromatography on anion-exchange (diethylaminoethyl- and triethylaminoethyl-)cellulose and Sephadex G-200 gel. 2. On ion-exchange cellulose columns both kinds of enzyme preparations yielded two major disaccharidase peaks. The first peak contained maltase Ia (=isomaltase) and maltase Ib (=invertase). The second peak contained maltase II and maltase III. 3. On Sephadex G-200 gel columns jejunal preparations yielded the corresponding peaks as on ion-exchange columns, but the peaks appeared in the reverse order in the effluent. The ileal preparation studied yielded a single peak on gel columns, containing all the activities studied and eluted with the `void volume'. 4. Precipitation with ethanol did not affect the behaviour of the enzymes during ion-exchange chromatography. When gel filtration was performed after ethanol precipitation of the enzymes, however, two peaks were obtained also with the ileal preparation, and subfractionation of the invertase was obtained with both kinds of preparations. 5. The second peak from ion-exchange chromatograms, containing maltase II and maltase III, on concentration was found to have very weak isomaltase activity, probably exerted by these enzymes as such. This activity accounts for only about 1% of the total isomaltase activity of the mucosa. 6. The results support the concept of the specificity of the human small-intestinal disaccharidases previously described after heat-inactivation experiments. The subfractionation of the invertase that under certain conditions is seen on Sephadex G-200 columns appears most likely to be an artifact. Consequently the nomenclature for the human maltose-, isomaltose- and sucrose-splitting enzymes proposed by another research group after gel-filtration chromatography studies should be abandoned. It seems more logical to keep the nomenclature based

  3. Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide

    PubMed Central

    Sano, Daisuke; Matsuo, Takahiro; Omura, Tatsuo

    2004-01-01

    The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H2N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology

  4. Characterization of Extracellular Proteins in Tomato Fruit using Lectin Affinity Chromatography and LC-MALDI-MS/MS analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The large-scale isolation and analysis of glycoproteins by lectin affinity chromatography coupled with mass spectrometry has become a powerful tool to monitor changes in the “glycoproteome” of mammalian cells. Thus far, however, this approach has not been used extensively for the analysis of plant g...

  5. Application of Pre-Column Labeling Liquid Chromatography for Canine Plasma-Free Amino Acid Analysis

    PubMed Central

    Azuma, Kazuo; Hirao, Yoshiko; Hayakawa, Yoshihiro; Murahata, Yusuke; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Okamoto, Yoshiharu; Ito, Norihiko

    2016-01-01

    Plasma-free amino acid (PFAA) levels are a useful metric for diagnosing cancer and providing a prognosis. However, the use of analysis of PFAA levels has been limited in the veterinary medicine field. We addressed the application of liquid chromatography (LC) using a pre-column labeling technique for analysis of canine PFAA levels. This method significantly shortened the analysis time relative to conventional methods. No diurnal fluctuations were detected at 9:00 AM in most PFAA levels, and food intake increased the levels of some PFAAs, including valine, leucine, tyrosine, phenylalanine, and proline. These results indicate that LC with pre-column labeling is useful for measuring canine PFAA levels, for which time of day and interval after food intake must be taken into consideration. PMID:26771650

  6. Preparation of a biomimetic polyphosphorylcholine monolithic column for immobilized artificial membrane chromatography.

    PubMed

    Zhao, XiangLong; Chen, WeiJia; Zhou, ZhengYin; Wang, QiQin; Liu, ZhengHua; Moaddel, Ruin; Jiang, ZhengJin

    2015-08-14

    The present work aims to prepare a novel phosphatidylcholine functionalized monolithic stationary phase by in situ co-polymerization of 12-methacryloyl dodecylphosphocholine (MDPC) and ethylene dimethacrylate (EDMA) for immobilized artificial membrane chromatography. Scanning electron microscopy, energy dispersive X-ray spectroscopy, FT-IR spectroscopy, pore size distribution analysis, ζ-potential analysis and micro-HPLC were used to evaluate the monolithic structure and physicochemical properties. Satisfactory morphology, high mechanical stability, good permeability and chromatographic performance were obtained on the optimized monolithic columns. A typical reverse-phase retention mechanism was observed over a wide range of organic solvent content (acetonitrile< 80%). The optimized poly(MDPC-co-EDMA) monolith exhibited good selectivity for proteins and basic drugs. Good correlation was observed between the retention on commercial IAM column (IAM.PC.DD2) and poly(MDPC-co-EDMA) monolith. This novel poly(MDPC-co-EDMA) monolith exhibited good potential for studying the drug-membrane interaction.

  7. Electrochemically-modulated liquid chromatography (EMLC): Column design, retention processes, and applications

    SciTech Connect

    Ting, E.Y.

    1997-10-08

    This work describes the continued development of a new separation technique, electrochemically-modulated liquid chromatography (EMLC), from column design, retention mechanisms to pharmaceutical applications. The introduction section provides a literature review of the technique as well as a brief overview of the research in each of the chapters. This section is followed by four chapters which investigate the issues of EMLC column design, the retention mechanism of monosubstituted aromatic compounds, and the EMLC-based applications to two important classes of pharmaceutical compounds (i.e., corticosteroids and benzodiazepines). These four sections have been removed to process separately for inclusion on the database. The dissertation concludes with a general summary, a prospectus, and a list of references cited in the General Introduction. 32 refs.

  8. Application of Pre-Column Labeling Liquid Chromatography for Canine Plasma-Free Amino Acid Analysis.

    PubMed

    Azuma, Kazuo; Hirao, Yoshiko; Hayakawa, Yoshihiro; Murahata, Yusuke; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Okamoto, Yoshiharu; Ito, Norihiko

    2016-01-01

    Plasma-free amino acid (PFAA) levels are a useful metric for diagnosing cancer and providing a prognosis. However, the use of analysis of PFAA levels has been limited in the veterinary medicine field. We addressed the application of liquid chromatography (LC) using a pre-column labeling technique for analysis of canine PFAA levels. This method significantly shortened the analysis time relative to conventional methods. No diurnal fluctuations were detected at 9:00 AM in most PFAA levels, and food intake increased the levels of some PFAAs, including valine, leucine, tyrosine, phenylalanine, and proline. These results indicate that LC with pre-column labeling is useful for measuring canine PFAA levels, for which time of day and interval after food intake must be taken into consideration. PMID:26771650

  9. Packed column supercritical fluid chromatography of sodium stearyl fumarate aqueous suspension.

    PubMed

    Gyllenhaal, Olle

    2006-03-01

    A method for the determination of sodium stearyl fumarate aqueous suspension is described. This straightforward method is based on homogenisation of the sample, dilution of a known aliquot with methanol to a suitable clear solution and mixing with an internal standard; (S)-naproxen. Separation and quantification is performed by packed column supercritical fluid chromatography on a commercial tartaric acid network polymeric column (tertbutylbenzoyl) with UV-detection at 214 nm. The precision of the presented method upon repeated analysis of a 20 mg/ml suspension is 0.5% (n = 8), and the yield is near 100%. Less than 5 min is required for the chromatographic separation with a resolution of about 3 to the internal standard. With some modification of the chromatographic conditions water samples can also be analysed. PMID:16174559

  10. Using the column wall itself as resistive heater for fast temperature gradients in liquid chromatography.

    PubMed

    De Pauw, Ruben; Pursch, Matthias; Desmet, Gert

    2015-11-13

    A new system is proposed for applying fast temperature gradients in liquid chromatography. It consists of a 0.7 mm × 150 mm fused-silica column coated with a 50 μm Nickel-layer, which is connecting with a power source and a temperature control system to perform fast and reproducible temperature gradients using the column wall itself as a resistive heater. Applying a current of 4A and passive cooling results in a maximal heating and cooling rate of, respectively, 71 and -21 °C/min. Multi-segment temperature gradients were superimposed on mobile phase gradients to enhance the selectivity for three sets of mixtures (pharmaceutical compounds, a highly complex mixture and an insecticide sample). This resulted in a higher peak count or better selectivities for the various mixtures.

  11. Kinetic efficiency of polar monolithic capillary columns in high-pressure gas chromatography.

    PubMed

    Kurganov, A A; Korolev, A A; Shiryaeva, V E; Popova, T P; Kanateva, A Yu

    2013-11-01

    Poppe plots were used for analysis of kinetic efficiency of monolithic sorbents synthesized in quartz capillaries for utilization in high-pressure gas chromatography. Values of theoretical plate time and maximum number of theoretical plates occurred to depend significantly on synthetic parameters such as relative amount of monomer in the initial polymerization mixture, temperature and polymerization time. Poppe plots let one to find synthesis conditions suitable either for high-speed separations or for maximal efficiency. It is shown that construction of kinetic Poppe curves using potential Van Deemter data demands compressibility of mobile phase to be taken into consideration in the case of gas chromatography. Model mixture of light hydrocarbons C1 to C4 was then used for investigation of influence of carrier gas nature on kinetic efficiency of polymeric monolithic columns. Minimal values of theoretical plate times were found for CO2 and N2O carrier gases.

  12. Extraction of antibiotic zwittermicin A from Bacillus thuringiensis by macroporous resin and silica gel column chromatography.

    PubMed

    Hao, Zaibin; Yan, Li; Liu, Jianguo; Song, Fuping; Zhang, Jie; Li, Xia

    2015-01-01

    To establish a production process capable of providing refined zwittermicin A (ZwA) on a large scale, the macroporous resin and silica gel column chromatography were used to separate and purify the antibiotic ZwA from the fermentation broth of Bacillus thuringiensis HD-1. The result of high-performance liquid chromatography-mass spectrometry after purification suggests that the samples of ZwA were of high purity, 89%, and the average yield was 20 mg L(-1). Erwinia herbicola LS005, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were used to assess the toxicity of ZwA. The antibiotic had strong antibacterial activity against E. herbicola LS005 and a color reaction with ninhydrin. PMID:25099664

  13. Multivariate data analysis to characterize gas chromatography columns for dioxin analysis.

    PubMed

    Do, Lan; Geladi, Paul; Haglund, Peter

    2014-06-20

    dipolar moment. Finally, the PCA and PLS analyses were complemented with linear regression analysis to identify the most orthogonal column combinations, which could be used in comprehensive two-dimensional gas chromatography (GC×GC) to enhance PCDD/F separation and congener profiling.

  14. Pressure programmed microbore column supercritical fluid chromatography-mass spectrometry for the determination of organophosphorus insecticides

    SciTech Connect

    Kalinoski, H.T.; Smith, R.D.

    1988-03-15

    The use of the high flow rate (HFR) interface for supercritical fluid chromatography-mass spectrometry (SFC-MS) is shown to allow operation under conditions which provide efficient pressure programmed separations with appropriate microbore (packed) HPLC columns. The combined advantages of selectivity offered by the microparticle-packed stationary phase and variable solvating power of the supercritical fluid are fully utilized in this approach. The greater sample loadings and lower detection limits possible using packed columns (based on concentration of sample in the injection solvent) compared with commercially available capillary columns are demonstrated for the determination of a series of organophosphorus insecticides. Low concentrations of polar fluid modifiers, generally required for high-quality separations in packed-column SFC, also function as mild chemical ionization reagents. Broad classes of thermally labile, higher molecular weight, moderately polar pesticides are amenable to identification by SFC-MS, which provides a sensitive, selective, and broadly applicable technique for the identification of pesticide compounds with detection limits in the part-per-billion range.

  15. Impact of reversed phase column pairs in comprehensive two-dimensional liquid chromatography.

    PubMed

    Allen, Robert C; Barnes, Brian B; Haidar Ahmad, Imad A; Filgueira, Marcelo R; Carr, Peter W

    2014-09-26

    A major issue in optimizing the resolving power of two-dimensional chromatographic separations is the choice of the two phases so as to maximize the distribution of the analytes over the separation space. In this work, we studied the choice of appropriate reversed phases to use in on-line comprehensive two-dimensional liquid chromatography (LC×LC). A set of four chemically different conventional bonded reversed phases was used in the first dimension. The second dimension column was either a conventional bonded C18 phase or a carbon-clad phase (CCP). The LC×LC chromatograms and contour plots were all rather similar indicating that the selectivities of the two phases were also similar regardless of the reverse phase column used in the first dimension. Further, the spatial coverage seen with all four first dimension stationary phases when paired with a second dimension C18 phase were low and the retention times were strongly correlated. However, when the C18 column was replaced with the CCP column much improved separations were observed with higher spatial coverages, greater orthogonalities and significant increases in the number of observed peaks.

  16. 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.

  17. Blind column selection protocol for two-dimensional high performance liquid chromatography.

    PubMed

    Burns, Niki K; Andrighetto, Luke M; Conlan, Xavier A; Purcell, Stuart D; Barnett, Neil W; Denning, Jacquie; Francis, Paul S; Stevenson, Paul G

    2016-07-01

    The selection of two orthogonal columns for two-dimensional high performance liquid chromatography (LC×LC) separation of natural product extracts can be a labour intensive and time consuming process and in many cases is an entirely trial-and-error approach. This paper introduces a blind optimisation method for column selection of a black box of constituent components. A data processing pipeline, created in the open source application OpenMS®, was developed to map the components within the mixture of equal mass across a library of HPLC columns; LC×LC separation space utilisation was compared by measuring the fractional surface coverage, fcoverage. It was found that for a test mixture from an opium poppy (Papaver somniferum) extract, the combination of diphenyl and C18 stationary phases provided a predicted fcoverage of 0.48 and was matched with an actual usage of 0.43. OpenMS®, in conjunction with algorithms designed in house, have allowed for a significantly quicker selection of two orthogonal columns, which have been optimised for a LC×LC separation of crude extractions of plant material. PMID:27154652

  18. Inverse gas chromatography. V - Computer simulation of diffusion processes on the column

    NASA Technical Reports Server (NTRS)

    Hattam, Paul; Munk, Petr

    1988-01-01

    The elution behavior of low molecular weight probes on inverse gas chromatography (IGC) columns is simulated using a computer. The IGC model is based on a polymer stationary phase of uniform thickness with a nonnegligible resitance to probe penetration. Three characteristic numbers are found to determine the whole process: Z(p) characterizing the distribution of the probe between phases, Z(f) describing the diffusion in the polymer phase, and Z(g) related to diffusion in the gaseous phase. For situations when Z(p)/Z(f) is less than 2, the standard evaluation procedures are virtually useless. The actual behavior of such systems is described.

  19. Analysis of isoamylase debranched starches with size exclusion chromatography utilizing PFG columns.

    PubMed

    Ciric, Jelena; Woortman, Albert J J; Loos, Katja

    2014-11-01

    Debranched starches were tested with a previously developed method for size exclusion chromatography (SEC) with multi detection utilizing different columns than usually used for the separation of starch in DMSO. A number of debranched starches were analyzed. This system allows good separation of amylose and amylopectin after debranching of starch, and provides quantitative information on the amylose content. Additionally molar mass versus hydrodynamic radii (Rh) distributions of various debranched starches show that the debranching was not 100% and that the differences in the structure of various starches can be followed. PMID:25129767

  20. Simple, specific analysis of organophosphorus and carbamate pesticides in sediments using column extraction and gas chromatography

    USGS Publications Warehouse

    Belisle, A.A.; Swineford, D.M.

    1988-01-01

    A simple, specific procedure was developed for the analysis of organophosphorus and carbamate pesticides in sediment. The wet soil was mixed with anhydrous sodium sulfate to bind water and the residues were column extracted in acetone:methylene chloride (1:l,v/v). Coextracted water was removed by additional sodium sulfate packed below the sample mixture. The eluate was concentrated and analyzed directly by capillary gas chromatography using phosphorus and nitrogen specific detectors. Recoveries averaged 93 % for sediments extracted shortly after spiking, but decreased significantly as the samples aged.

  1. Investigation of the heterogeneity of heterogalactan from the fruit bodies of Fomitopsis pinicola, by employing concanavalin A-Sepharose affinity chromatography.

    PubMed

    Usui, T; Hosokawa, S; Mizuno, T; Suzuki, T; Meguro, H

    1981-04-01

    A heterogalactan was isolated from the hot water extract of fruit bodies of Fomitopsis pinicola by a combination of fractionation procedures including precipitation with ethanol and with Cetavlon, and chromatography on columns of DEAE-cellulose and Sephadex G-100. Despite its apparent homogeneity on gel filtration, zone electrophoresis, sedimentation equilibration, and immunodiffusion analyses, the neutral component of heterogalactan was further fractionated into unbound, weakly bound, and strongly bound forms by affinity chromatography on a column of concanavalin A-Sepharose CL 4B. The former two polysaccharides fractions eluted with 0.1 M phosphate buffer (pH 7.0) were found to be a fucogalactan and a mannofucogalactan, respectively. A more tightly bound fraction (mannofucogalactan) was subsequently eluted with 0.1 M glucose in 1 M NaCl. The results of methylation, complete Smith degradation, and proton and 13C NMR spectroscopic analyses indicated that the three kinds of heterogalactans are all highly branched polysaccharides containing a framework of (1 leads to 6)-linked alpha-D-galactopyranosyl residues, the C-2 positions of which are substituted in different proportions with either single L-fucopyranosyl residues or disaccharide units of 3-O-alpha-D-mannopyranosyl-L-fucopyranose residues.

  2. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  3. Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins.

    PubMed

    Ito, Y; Yamasaki, Y; Seno, N; Matsumoto, I

    1986-04-01

    Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin. PMID:3711062

  4. Comparing multistep immobilized metal affinity chromatography and multistep TiO2 methods for phosphopeptide enrichment.

    PubMed

    Yue, Xiaoshan; Schunter, Alissa; Hummon, Amanda B

    2015-09-01

    Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multistep enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multiphosphopeptides as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multistep enrichment. PMID:26237447

  5. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    PubMed

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

  6. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

    PubMed Central

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

  7. 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.

  8. 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. PMID:26573171

  9. Simulation of the dynamic packing behavior of preparative chromatography columns via discrete particle modeling.

    PubMed

    Dorn, Martin; Hekmat, Dariusch

    2016-03-01

    Preparative packed-bed chromatography using polymer-based, compressible, porous resins is a powerful method for purification of macromolecular bioproducts. During operation, a complex, hysteretic, thus, history-dependent packed bed behavior is often observed but theoretical understanding of the causes is limited. Therefore, a rigorous modeling approach of the chromatography column on the particle scale has been made which takes into account interparticle micromechanics and fluid-particle interactions for the first time. A three-dimensional deterministic model was created by applying Computational Fluid Dynamics (CFD) coupled with the Discrete Element Method (DEM). The column packing behavior during either flow or mechanical compression was investigated in-silico and in laboratory experiments. A pronounced axial compression-relaxation profile was identified that differed for both compression strategies. Void spaces were clearly visible in the packed bed after compression. It was assumed that the observed bed inhomogeneity was because of a force-chain network at the particle scale. The simulation satisfactorily reproduced the measured behavior regarding packing compression as well as pressure-flow dependency. Furthermore, the particle Young's modulus and particle-wall friction as well as interparticle friction were identified as crucial parameters affecting packing dynamics. It was concluded that compaction of the chromatographic bed is rather because of particle rearrangement than particle deformation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:363-371, 2016.

  10. Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine.

    PubMed Central

    Smith, G D; Roberts, D V; Daday, A

    1977-01-01

    Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo. PMID:889568

  11. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

    PubMed

    Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

    2015-01-01

    A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.

  12. Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography.

    PubMed

    Kuhar, Kalika; Mittal, Anuradha; Kansal, Rekha; Gupta, Vijay Kumar

    2014-02-01

    Plant protease inhibitors (PIs) are generally small proteins which play key roles in regulation of endogenous proteases and may exhibit antifeedant, antifungal, antitumor and cytokine inducing activities. Dolichos biflorus (horse gram) is an unexploited legume, which is rich in nutrients and also has therapeutic importance. It contains a double-headed PI, which is an anti-nutritional factor. As there is no report available on its simultaneous removal and purification in single step, in this study, a double-headed PI active against both trypsin and chymotrypsin was purified from Dolichos biflorus to -14-fold with -84% recovery using an immobilized metal affinity chromatography (IMAC) medium consisting of Zn-alginate beads. The method was single-step, fast, simple, reliable and economical. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH range of 2.0-12.0 and up to a temperature of 100 degrees C for 20 min. The optimum temperature for trypsin and chymotrypsin inhibitor was observed to be 50 degrees C and 37 degrees C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC using Zn-alginate beads was useful in simultaneous purification and removal of an anti-nutritional factor from horse gram flour in single step. This procedure may also be employed for purification of other plant PIs in one step.

  13. Ligand affinity chromatography, an indispensable method for the purification of soluble cytokine receptors and binding proteins.

    PubMed

    Novick, Daniela; Rubinstein, Menachem

    2012-01-01

    Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity. PMID:22131033

  14. Analysis of the Glycoproteome of Toxoplasma gondii using Lectin Affinity Chromatography and Tandem Mass Spectrometry

    PubMed Central

    Luo, Qilie; Upadhya, Rajendra; Zhang, Hong; Madrid-Aliste, Carlos; Nieves, Edward; Kim, Kami; Angeletti, Ruth Hogue; Weiss, Louis M.

    2011-01-01

    Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1 to 5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This is data provides a large scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates. PMID:21920448

  15. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography

    PubMed Central

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S.

    2015-01-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  16. Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin

    PubMed Central

    Liang, Aiye; Thakkar, Jay N.; Hindle, Michael; Desai, Umesh R.

    2013-01-01

    Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I35, I55 and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I35 possessed good anticoagulation potential (APTT = 4.2 μM; PT = 6.8 μM), while I55 and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4 m/z, which most probably arise from variably functionalized (β-O4—β-β-linked trimers and/or a β-O4—β-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants. PMID:23122400

  17. Analysis of Lidocaine Interactions with Serum Proteins Using High-Performance Affinity Chromatography

    PubMed Central

    Soman, Sony; Yoo, Michelle J.; Jang, Yoon Jeong; Hage, David S.

    2010-01-01

    High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α1–acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (Ka) of 1.1-1.7 × 105 M-1 at 37 °C and pH 7.4. Lidocaine had weak-to-moderate binding to HSA, with a Ka in the range of 103 to 104 M-1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes. PMID:20138813

  18. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

    PubMed Central

    Choi, Ryan; Kelley, Angela; Leibly, David; Nakazawa Hewitt, Stephen; Napuli, Alberto; Van Voorhis, Wesley

    2011-01-01

    The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure. PMID:21904040

  19. Characterization of glycoproteins in pancreatic cyst fluid using a high performance multiple lectin affinity chromatography platform

    PubMed Central

    Gbormittah, Francisca Owusu; Haab, Brian B.; Partyka, Katie; Garcia-Ott, Carolina; Hancapie, Marina; Hancock, William S.

    2014-01-01

    Currently, pancreatic cancer is the fourth cause of cancer death. In 2013, it is estimated that approximately 38,460 people will die of pancreatic cancer. Early detection of malignant cyst (pancreatic cancer precursor) is necessary to help prevent late diagnosis of the tumor. In this study, we characterized glycoproteins and non-glycoproteins on pooled mucinous (n=10) and non-mucinous (n=10) pancreatic cyst fluid to identify ‘proteins of interest’ to differentiate between mucinous cyst from non-mucinous cyst and investigate these proteins as potential biomarker targets. An automated multi-lectin affinity chromatography (M-LAC) platform was utilized for glycoprotein enrichment followed by nano-LC-MS/MS analysis. Spectral count quantitation allowed for the identification of proteins with significant differential levels in mucinous cysts from non-mucinous cysts of which one protein (periostin) was confirmed via immunoblotting. To exhaustively evaluate differentially expressed proteins, we used a number of proteomic tools including; gene ontology classification, pathway and network analysis, Novoseek data mining and chromosome gene mapping. Utilization of complementary proteomic tools, revealed that several of the proteins such as mucin 6 (MUC6), bile salt-activated lipase (CEL) and pyruvate kinase lysozyme M1/M2 with significant differential expression have strong association with pancreatic cancer. Further, chromosome gene mapping demonstrated co-expressions and co-localization of some proteins of interest including 14-3-3 protein epsilon (YWHAE), pigment epithelium derived factor (SERPINF1) and oncogene p53. PMID:24303806

  20. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S

    2014-08-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  1. The Lectin Frontier Database (LfDB), and data generation based on frontal affinity chromatography.

    PubMed

    Hirabayashi, Jun; Tateno, Hiroaki; Shikanai, Toshihide; Aoki-Kinoshita, Kiyoko F; Narimatsu, Hisashi

    2015-01-01

    Lectins are a large group of carbohydrate-binding proteins, having been shown to comprise at least 48 protein scaffolds or protein family entries. They occur ubiquitously in living organisms-from humans to microorganisms, including viruses-and while their functions are yet to be fully elucidated, their main underlying actions are thought to mediate cell-cell and cell-glycoconjugate interactions, which play important roles in an extensive range of biological processes. The basic feature of each lectin's function resides in its specific sugar-binding properties. In this regard, it is beneficial for researchers to have access to fundamental information about the detailed oligosaccharide specificities of diverse lectins. In this review, the authors describe a publicly available lectin database named "Lectin frontier DataBase (LfDB)", which undertakes the continuous publication and updating of comprehensive data for lectin-standard oligosaccharide interactions in terms of dissociation constants (Kd's). For Kd determination, an advanced system of frontal affinity chromatography (FAC) is used, with which quantitative datasets of interactions between immobilized lectins and >100 fluorescently labeled standard glycans have been generated. The FAC system is unique in its clear principle, simple procedure and high sensitivity, with an increasing number (>67) of associated publications that attest to its reliability. Thus, LfDB, is expected to play an essential role in lectin research, not only in basic but also in applied fields of glycoscience. PMID:25580689

  2. [Ion-pair chromatography-indirect ultraviolet detection for determination of tetraethyl ammonium using a monolithic column and a packed column].

    PubMed

    Zou, Chunmiao; Zhang, Xiaodong; Yu, Hong; Guan, Chao; Wang, Miaoyu

    2015-07-01

    Two methods were developed for the determination of tetraethyl ammonium by ion-pair chromatography-indirect ultraviolet detection using a monolithic column and a packed column with ionic liquid as additive in mobile phase. Chromatographic separations were performed on a monolithic column and a packed column both on reversed phase using imidazolium ionic liquid aqueous solution-ion-pair reagent-organic solvent as mobile phase. The effects of the background ultraviolet absorption reagent, detection wavelength, ion-pair reagent, organic solvent, column temperature and flow rate on the determination of tetraethyl ammonium were investigated. The difference between the two chromatographic columns was compared and the retention rules were discussed. Under the optimized chromatographic conditions, for tetraethyl ammonium on monolithic column and packed column, the retention times were 2.40 and 3.02 min; the detection limits (S/N=3), 0.04 and 0.07 mg/L; the RSDs (n = 5) for peak areas, 0.16% and 0.11%; and the RSDs (n=5) for retention times, 0.02% and 0.01%, respectively. The two methods have been successfully applied to the determination of tetraethyl ammonium ionic liquids synthesized by laboratory. The recoveries of the tetraethyl ammonium after spiking were 98.2% and 99.1%, respectively. The two methods can meet the requirements for the quantitative analysis of tetraethyl ammonium.

  3. Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

    PubMed

    Jiang, Han-Peng; Chu, Jie-Mei; Lan, Meng-Dan; Liu, Ping; Yang, Na; Zheng, Fang; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-09-01

    More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two

  4. Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

    PubMed

    Jiang, Han-Peng; Chu, Jie-Mei; Lan, Meng-Dan; Liu, Ping; Yang, Na; Zheng, Fang; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-09-01

    More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two

  5. Separation of uremic toxins from urine with resorcinarene-based ion chromatography columns.

    PubMed

    Panahi, Tayyebeh; Weaver, Douglas J; Lamb, John D; Harrison, Roger G

    2015-01-01

    People with chronic kidney disease suffer from uremic toxins which accumulate in their bodies. Detection and quantification of uremic toxins help diagnose kidney problems and start patient care. The aim of this research was to seek a new method to assist this diagnosis by trace level detection and separation of guanidine containing uremic toxins in water and urine. To detect and quantify the uremic toxins, new stationary phases for ion chromatography (IC) columns based on glutamic acid functionalized resorcinarenes bound to divinylbenzene macroporous resin were prepared. The new column packing material afforded separation of the five compounds: guanidinoacetic acid, guanidine, methylguanidine, creatinine, and guanidinobenzoic acid in 30min. Peak resolutions ranged from 7.6 to 1.3. Gradient elutions at ambient temperature with methanesulfonic acid (MSA) solution as eluent resulted in detection levels in water from 10 to 47ppb and in synthetic urine from 28 to 180ppb. Limits of quantification for the analytes using pulsed amperometric detection were 30-160ppb in water and 93-590ppb in urine. Trace levels of creatinine (1ppm) were detected in the urine of a healthy individual using the columns.

  6. Retention behavior of hydrophobic organic chemicals as a function of temperature in soil leaching column chromatography.

    PubMed

    Liang, Xinmiao; Xu, Feng; Lin, Bingcheng; Su, Fan; Schramm, Karl-Werner; Kettrup, Antonius

    2002-11-01

    To study the transport mechanism of hydrophobic organic chemicals (HOCs) and the energy change in soil/solvent system, a soil leaching column chromatographic (SLCC) experiment at an environmental temperature range of 20-40 degrees C was carried out, which utilized a reference soil (SP 14696) packed column and a methanol-water (1:4 by volume ratio) eluent. The transport process quickens with the increase of column temperature. The ratio of retention factors at 30 and 40 degrees C (k'30/k'40) ranged from 1.08 to 1.36. The lower enthalpy change of the solute transfer in SLCC (from eluent to soil) than in conventional reversed-phase liquid chromatography (e.g., from eluent to C18) is consistent with the hypothesis that HOCs were dominantly and physically partitioned between solvent and soil. The results were also verified by the linear solvation energy relationships analysis. The chief factor controlling the retention was found to be the solute solvophobic partition, and the second important factor was the solute hydrogen-bond basicity, while the least important factors were the solute polarizability-dipolarity and hydrogen-bond acidity. With the increase of temperature, the contributions of the solute solvophobic partition and hydrogen-bond basicity gradually decrease, and the latter decreases faster than the former. PMID:12430644

  7. Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics.

    PubMed Central

    Wright, C L; Warsy, A S; Holroyde, M J; Trayer, I P

    1978-01-01

    The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity-chromatography

  8. Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps.

    PubMed

    Roberts, Peter L

    2014-01-01

    The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG-1 & -3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion-exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non-enveloped viruses over the life-time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion-exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X-100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process.

  9. [Online enrichment ability of restricted-access column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride].

    PubMed

    Zhang, Xiaohui; Wang, Rong; Xie, Hua; Yin, Qiang; Li, Xiaoyun; Jia, Zhengping; Wu, Xiaoyu; Zhang, Juanhong; Li, Wenbin

    2013-05-01

    The online enrichment ability of the restricted-access media (RAM) column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride in plasma was studied. The RAM-HPLC system consisted of an RAM column as enrichment column and a C18 column as analytical column coupled via the column switching technique. The effects of the injection volume on the peak area and the systematic pressure were studied. When the injection volume was less than 100 microL, the peak area increased with the increase of the injection volume. However, when the injection volume was more than 80 microL, the pressure of whole system increased obviously. In order to protect the whole system, 80 microL was chosen as the maximum injection volume. The peak areas of ordinary injection and the large volume injection showed a good linear relationship. The enrichment ability of RAM-HPLC system was satisfactory. The system was successfully used for the separation and detection of the trace benazepril hydrochloride in rat plasma after its administration. The sensitivity of HPLC can be improved by RAM pre-enrichment. It is a simple and economic measurement method.

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

    PubMed

    Gritti, Fabrice; McDonald, Thomas; Gilar, Martin

    2016-06-17

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

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

    PubMed

    Gritti, Fabrice; McDonald, Thomas; Gilar, Martin

    2016-06-17

    250μm×100mm fused silica glass capillaries were packed with 1.8μm high-strength silica (HSS) fully porous particles. They were prepared without bulky stainless steel endfittings and metal frits, which both generate significant sample dispersion. The isocratic efficiencies and gradient peak capacities of these prototype capillary columns were measured for small molecules (n-alkanophenones) using a home-made ultra-low dispersive micro-HPLC instrument. Their resolution power was compared to that of standard 2.1mm×100mm very high-pressure liquid chromatography (vHPLC) narrow-bore columns packed with the same particles. The results show that, for the same column efficiency (25000 plates) and gradient steepness (0.04min(-1)), the peak capacity of the 250μm i.d. capillary columns is systematically 15-20% higher than that of the 2.1mm i.d. narrow-bore columns. A validated model of gradient chromatography enabled one to predict accurately the observed peak capacities of the capillary columns for non-linear solvation strength retention behavior and under isothermal conditions. Thermodynamics applied to the eluent quantified the temperature difference for the thermal gradients in both capillary and narrow-bore columns. Experimental data revealed that the gradient peak capacity is more affected by viscous heating than the column efficiency. Unlike across 2.1mm i.d. columns, the changes in eluent composition across the 250μm i.d. columns during the gradient is rapidly relaxed by transverse dispersion. The combination of (1) the absence of viscous heating and (2) the high uniformity of the eluent composition across the diameter of capillary columns explains the intrinsic advantage of capillary over narrow-bore columns in gradient vHPLC. PMID:27185055

  12. Selection of ceramic fluorapatite-binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography.

    PubMed

    Islam, Tuhidul; Bibi, Noor Shad; Vennapusa, Rami Reddy; Fernandez-Lahore, Marcelo

    2013-08-01

    Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA-specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N-terminal sequence was found in two selected peptides: F4-2 (KPRSMLH) and F5-4 (KPRSVSG). The peptide F5-4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5-4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage-derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins.

  13. Residual on column host cell protein analysis during lifetime studies of protein A chromatography.

    PubMed

    Lintern, Katherine; Pathak, Mili; Smales, C Mark; Howland, Kevin; Rathore, Anurag; Bracewell, Daniel G

    2016-08-26

    Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC-MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number. PMID:27473513

  14. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

    PubMed Central

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-01-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  15. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-15

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

  16. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  17. Glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides

    PubMed Central

    Alvarez-Manilla, Gerardo; Warren, Nicole L.; Atwood, James; Orlando, Ron; Dalton, Stephen; Pierce, Michael

    2011-01-01

    Numerous studies have recently focused on the identification of specific glycan biomarkers; given the important roles that protein linked glycans play, for example, during development and disease progression. The identification of protein glycobiomarkers, which are part of a very complex proteome, has involved the use of fractionation techniques such as lectin affinity chromatography. In this study, the glycoproteomic characterization of pluripotent murine embryonic stem cells (ES) and from ES cells that were differentiated into embroid bodies (EB) was performed using immobilized Concanavalin A (ConA). This procedure allowed the isolation of glycopeptides that express biantennary and hybrid N-linked structures (ConA2 fraction) as well as high mannose glycans (ConA3 fraction), that were abundant in both ES and EB stages. A total of 293 unique N-linked glycopeptide sequences (from 180 glycoproteins) were identified in the combined data sets from ES and EB cells. Of these glycopeptides, a total of 119 sequences were identified exclusively in only one of the lectin bound fractions, (24 in the ES-ConA2, 15 in the ES-ConA3, 16 in the EB-ConA2 and 64 in the EB-ConA3). Results from this study allowed the identification of individual N-glycosylation sites of proteins that express specific glycan types. The absence of some of these lectin bound glycopeptides in a cell stage suggested that they were derived from proteins that were either expressed exclusively on a defined developmental stage, or were expressed in both cell stages but carried the lectin bound oligosaccharides in only one of them. Therefore, these lectin bound glycopeptides can be considered as stage specific glycobiomarkers. PMID:19545112

  18. Separation of recombinant human protein C from transgenic animal milk using immobilized metal affinity chromatography.

    PubMed

    Dalton, J C; Bruley, D F; Kang, K A; Drohan, W N

    1997-01-01

    Protein C is an important serine protease due to its ability to proteolytically cleave activated Factors V and VIII. Excess coagulation and blood agglutination can lead to plugged capillaries, thereby reducing oxygen transport to interstitial tissues. To treat patients with hereditary and acquired protein C deficiency would require a greater amount of Protein C than that available from human plasma. However, the potential demand for this protein could be met by the production of human protein C from transgenic animal mammary glands. Thus, research into inexpensive, efficient methods to purify proteins from transgenic animal milk will be a critical area of study for the large scale production of protein C. Immobilized metal affinity chromatography (IMAC) is a novel method for the purification of protein C. A proposed method of purification is to take advantage of protein C's strong metal ion binding characteristics with IMAC to assist in the separation from transgenic animal milk. The separation procedure is benchmarked against current systems in use by the American Red Cross for purification of Protein C from transgenic porcine milk. Common problems in developing separation schemes for new therapeutics are the initial availability of the product (protein), and time-to-market concerns. Extensive experimental tests for scaleable purification schemes are often cost and time prohibitive. In order to optimize an IMAC protocol with minimal waste of time and resources, total quality management tools have been adopted. Initial experiments were designed to choose buffer conditions, eluents, immobilized valence metals, and flow rates using Taguchi experimental design, which is a total quality management (TQM) tool. One of the values of Taguchi methods lies in the use of Latin orthogonal sets. Through the use of the orthogonal sets, the total number of experiments may be reduced, shortening the focus time on optimal conditions.

  19. Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography.

    PubMed

    Tang, Weizhuo; Sun, Baoshan; Zhao, Yuqing

    2014-02-01

    In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil production, was achieved using combined column chromatography over macroporous and polyamide resins. To optimize the RA enrichment process, the performance and separation characteristics of nine selected macroporous resins with different chemical and physical properties were investigated. SP825 resin was the most effective: the content of RA increased from 0.27% in the original extract to 16.58% in the 50% ethanol fraction (a 61.4-fold increase). During further purification treatment on polyamide resin, 90.23% pure RA could be obtained in the 70% ethanol fraction. RA with a higher purity (>95%) could also be easily obtained using one crystallization operation. The proposed method is simple, easily operated, cost-effective, and environmentally friendly and is suitable for both large-scale RA production and waste management.

  20. New thermionic detector utilizing orthogonal nebulization for capillary column liquid chromatography.

    PubMed

    Gluckman, J C; Novotny, M

    1984-11-30

    A new version of the previously studied phosphorus-sensitive, dual-flame thermionic detector has been developed for microcolumn liquid chromatography. The total column effluent is orthogonally nebulized and aspirated directly into a primary air-hydrogen diffusion flame. Phosphorus compounds with molecular weights extending beyond 500 a.m.u. are then selectively detected by measuring the conductivity of the secondary flame in the presence of a rubidium silicate bead. The sensitivity was found to be 1.36 X 10(-11) g phosphorus/sec at the maximum of a Gaussian peak, and the signal increased linearly with concentration over 2 orders of magnitude for dilute samples. Possible mechanisms accounting for the negative orientation of the signal and for the limited dynamic range are discussed.

  1. Separation of Be and Al for AMS using single-step column chromatography

    NASA Astrophysics Data System (ADS)

    Binnie, Steven A.; Dunai, Tibor J.; Voronina, Elena; Goral, Tomasz; Heinze, Stefan; Dewald, Alfred

    2015-10-01

    With the aim of simplifying AMS target preparation procedures for TCN measurements we tested a new extraction chromatography approach which couples an anion exchange resin (WBEC) to a chelating resin (Beryllium resin) to separate Be and Al from dissolved quartz samples. Results show that WBEC-Beryllium resin stacks can be used to provide high purity Be and Al separations using a combination of hydrochloric/oxalic and nitric acid elutions. 10Be and 26Al concentrations from quartz samples prepared using more standard procedures are compared with results from replicate samples prepared using the coupled WBEC-Beryllium resin approach and show good agreement. The new column procedure is performed in a single step, reducing sample preparation times relative to more traditional methods of TCN target production.

  2. Extra-column dispersion of macromolecular solutes in aqueous-phase size-exclusion chromatography.

    PubMed

    Grznárová, G; Polakovic, M; Acai, P; Görner, T

    2004-06-18

    A set of dextran standards was used to study the extra-column dispersion in conventional chromatographic equipment at a broad range of molecular weights, different mobile phase flow rates and connecting tube lengths and diameters. All known correlations for the tube dispersion at laminar flow, including those for short tubes, overestimated the values of the variance of the outlet concentration signal. The difference increased with the solute molecular weight and the flow rate. It was assumed that the discrepancy was due to the effect of natural convection invoked by the density differences of the injected dextran solutions and water. A suitable approximation of the relative band spreading was suggested in a form of a power function of the Reynolds and Schmidt numbers. A significant decrease of the dispersion was observed when the chromatography tubing was coiled into a circle. This decrease was successfully predicted combining the existing correlations for long coiled tubes and short straight tubes.

  3. Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography.

    PubMed

    Tang, Weizhuo; Sun, Baoshan; Zhao, Yuqing

    2014-02-01

    In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil production, was achieved using combined column chromatography over macroporous and polyamide resins. To optimize the RA enrichment process, the performance and separation characteristics of nine selected macroporous resins with different chemical and physical properties were investigated. SP825 resin was the most effective: the content of RA increased from 0.27% in the original extract to 16.58% in the 50% ethanol fraction (a 61.4-fold increase). During further purification treatment on polyamide resin, 90.23% pure RA could be obtained in the 70% ethanol fraction. RA with a higher purity (>95%) could also be easily obtained using one crystallization operation. The proposed method is simple, easily operated, cost-effective, and environmentally friendly and is suitable for both large-scale RA production and waste management. PMID:24381020

  4. Temperature-assisted on-column solute focusing: a general method to reduce pre-column dispersion in capillary high performance liquid chromatography.

    PubMed

    Groskreutz, Stephen R; Weber, Stephen G

    2014-08-01

    Solvent-based on-column focusing is a powerful and well known approach for reducing the impact of pre-column dispersion in liquid chromatography. Here we describe an orthogonal temperature-based approach to focusing called temperature-assisted on-column solute focusing (TASF). TASF is founded on the same principles as the more commonly used solvent-based method wherein transient conditions are created that lead to high solute retention at the column inlet. Combining the low thermal mass of capillary columns and the temperature dependence of solute retention TASF is used effectively to compress injection bands at the head of the column through the transient reduction in column temperature to 5°C for a defined 7mm segment of a 6cm long 150μm I.D. column. Following the 30s focusing time, the column temperature is increased rapidly to the separation temperature of 60°C releasing the focused band of analytes. We developed a model to simulate TASF separations based on solute retention enthalpies, focusing temperature, focusing time, and column parameters. This model guides the systematic study of the influence of sample injection volume on column performance. All samples have solvent compositions matching the mobile phase. Over the 45-1050nL injection volume range evaluated, TASF reduces the peak width for all solutes with k' greater than or equal to 2.5, relative to controls. Peak widths resulting from injection volumes up to 1.3 times the column fluid volume with TASF are less than 5% larger than peak widths from a 45nL injection without TASF (0.07 times the column liquid volume). The TASF approach reduced concentration detection limits by a factor of 12.5 relative to a small volume injection for low concentration samples. TASF is orthogonal to the solvent focusing method. Thus, it can be used where on-column focusing is required, but where implementation of solvent-based focusing is difficult.

  5. Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography.

    PubMed Central

    Callaghan, J M; Toh, B H; Simpson, R J; Baldwin, G S; Gleeson, P A

    1992-01-01

    We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized

  6. Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrices

    SciTech Connect

    Swaisgood, H.E.; Chaiken, I.M.

    1986-07-01

    Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. The self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatograhic column containing immobilized neurophysin predominantly in the monomer form. Both (/sup 3/H)(Arg/sup 8/)vasopressin (AVP) and /sup 125/I-BNP II were rapidly eluted (<25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k..sqrt../sub 3/, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart. This latter difference may reflect the probability of rebinding to contiguous sites immobilized on a surface, a feature which would be related to that for contiguous sites on a membrane.

  7. Effect of pre- and post-column band broadening on the performance of high-speed chromatography columns under isocratic and gradient conditions.

    PubMed

    Vanderlinden, Kim; Broeckhoven, Ken; Vanderheyden, Yoachim; Desmet, Gert

    2016-04-15

    We report on the results of an experimental and theoretical study of the effect of the extra-column band broadening (ECBB) on the performance of narrow-bore columns filled with the smallest particles that are currently commercially available. Emphasis is on the difference between the effect of ECBB under gradient and isocratic conditions, as well as on the ability to model and predict the ECBB effects using well-established band broadening expressions available from the theory of chromatography. The fine details and assumptions that need to be taken into account when using these expressions are discussed. The experiments showed that, the steeper the gradient, the more pronounced the extra-column band broadening losses become. Whereas the pre-column band broadening can in both isocratic and gradient elution be avoided by playing on the possibilities to focus the analytes on top of the column (e.g. by using the POISe injection method when running isocratic separations), the post-column extra-column band broadening is inescapable in both cases. Inducing extra-column band broadening by changing the inner diameter of the post-column tubing from 65 to 250 μm, we found that all peaks in the chromatogram are strongly affected (around a factor of 1.9 increase in relative peak width) when running steep gradients, while usually only the first eluting peak was affected in the isocratic mode or when running shallow gradients (factor 1.6-1.8 increase in relative peak width for the first eluting analyte).

  8. Rapid separation of polysaccharides using a novel spiral coil column by high-speed countercurrent chromatography.

    PubMed

    Li, Weili; Wu, Tao

    2016-04-01

    The separation of polysaccharides is time consuming. We developed and optimized a type-J counter-current chromatography system with a novel tri-rotor spiral coil column for the rapid separation of polysaccharides. The optimal composition of an aqueous PEG1000/K2 HPO4 /KH2 PO4 system was found to be 14:16:14 w/w/w where the lower phase was the mobile phase. Optimal performance was achieved at a column rotational speed, temperature, and flow rate of 1200 rpm, 45°C, and 3.0 mL/min, respectively. The mobile phase was pumped from the inner terminal in a ''head-to-tail'' elution mode. Polysaccharide LCP-1 (10.7 mg) was successfully obtained in high purity in one step from 50.0 mg of a crude polysaccharide extracted from the lychee fruit (Litchi chinensis) within 100 min. LCP-1 possess a number-average molecular weight and weight-average molecular weight of 1.05 × 10(5) and 1.59 × 10(5) kDa, respectively. The monosaccharide composition consists of the molar ratio of glucose, galactose, and arabinose of 1.3:3.5:1.

  9. Determination of trace benzene hydrocarbons in Jiaozhou Bay by enrichment and capillary column gas chromatography

    NASA Astrophysics Data System (ADS)

    Cui, Xian-Zhou; Cui, Hang

    1996-06-01

    Trace amounts of benzene hydrocarbons obtained in Jiaozhou Bay (Qingdao) were enriched by sorption on a GDX-102 column and eluted by carbon disulfide. The eluate was concentrated and then determined by capillary column gas chromatography. The contents of various kinds of benzene hydrocarbons in Jiaozhou Bay coastal water were benzene (22.3 141.6)×10-9 g/L; toluate (15.2 94.0)×10-9 g/L; ethyl benzene (11.8 85.1)×10-9 g/L; p—xylene (15.2 78.5)×10-9 g/L; m—xylene (10.9 79.4)×10-9 g/L; o—xylene (12.4 80.1)×10-9 g/L; iso—propyl (8.4 73.1)×10-9 g/L; n—propyl (6.9 76.4)×10-9 g/L; 1, 3, 5-trimethylbenzene (10.9 35.9)×10-9 g/L; 1, 2, 4-trimethybenzene (10.0 38.0)×10-9 g/L; n—butybenzene (8.1 34.6)×10-9 g/L. The recovery of benzene hydrocarbons was (85.1 95.6)%.

  10. Optimization of preparative separation and purification of total polyphenols from Sargassum tenerrimum by column chromatography

    NASA Astrophysics Data System (ADS)

    Haider, Samee; Li, Zhenxing; Lin, Hong; Jamil, Khalid

    2009-12-01

    Polyphenols from the ethanol extracts of Sargassum tenerrimum (ST) with potent antiallergic effects were studied to optimize separation process through column chromatography. The adsorption and desorption characteristics of three widely used adsorbents: macroporous resin, silica gel, and polyvinylpolypyrrolidone (PVPP), were critically evaluated respectively and studied for the optimization of preparative separation of polyphenols. Static operations on these adsorbents showed that macroporous resin had the best adsorption and desorption capability among the three adsorbents. Dynamic adsorption and desorption with macroporous resin packed column were also conducted to optimize the parameters such as: with the optimal values shown in brackets, the concentration of extract solution (4 times diluted), pH value (6-7), adsorption speed (3 BV h-1, bed volumes/per hour), concentration of ethanol (80%), elution speed (3 BV h-1) and elution volume (7 BV). The chromatographic process so optimized gave a purity of 62.43% from the crude polyphenols, providing a promising basis for large scale preparation of bioactive polyphenols upon further scaling up tests.

  11. Understanding and diminishing the extra-column band broadening effects in supercritical fluid chromatography.

    PubMed

    De Pauw, Ruben; Shoykhet Choikhet, Konstantin; Desmet, Gert; Broeckhoven, Ken

    2015-07-17

    Supercritical fluid chromatography, where a low-viscosity mobile phase such as carbon dioxide is used, proves to be an excellent technique for fast and efficient separations, especially when sub-2μm particles are used. However, to achieve high velocities when using these small particles, and in order to stay within the flow rate range of current SFC-instruments, narrow columns (e.g. 2.1mm ID) must be used. Unfortunately, state-of-the-art instrumentation is limiting the full separation power of these narrower columns due to significant extra-column band broadening effects. The present work identifies and quantifies the different contributions to extra-column band broadening in SFC such as the influence of the sample solvent, injection volume, extra-column volumes and detector cell volume/design. When matching the sample solvent to the mobile phase in terms of elution strength and polarity (e.g. using hexane/ethanol/isopropanol 85/10/5vol%) and lowering the injection volume to 0.4μL, the plate count can be increased from 7600 to 21,300 for a low-retaining compound (k'=2.3) on a 2.1mm×150mm column (packed with 1.8μm particles). The application of a water/acetonitrile mixture as sample solvent was also investigated. It was found that when the volumetric ratio of water/acetonitrile was optimized, only a slightly lower plate count was measured compared to the hexane-based solvent when minimizing injection and extra-column volume. This confirms earlier results that water/acetonitrile can be used if water-soluble samples are considered or when a less volatile solvent is preferred. Minimizing the ID of the connection capillaries from 250 to 65μm, however, gives no further improvement in obtained efficiency for early-eluting compounds when a standard system configuration with optimized sample solvent was used. When switching to a state-of-the-art detector design with reduced (dispersion) volume (1.7-0.6μL), an increase in plate count is observed (from 11,000 to 14

  12. [Determination of anions in recycle sodium formate from sodium hydrosulfite industry with single column ion chromatography].

    PubMed

    Feng, L; Ding, M

    1999-07-01

    The analysis of recycle materials from industrial waste water of sodium hydrosulfite production was made by single-column anion exchange chromatography. The recycle material was sodium formate (HCOONa) in which various anions, such as SO(3)2-, Cl-, thiosulfate, hydroxyethyl sulfonate (HOC2H4SO3-), hydroxyethyl thiosulfate (HOC2H4S2O3-), SO(4)2- and NO3- as impurities were existed simultaneously. Except hydroxyethyl sulfonate anion, all other ions can be separated by using anion exchange column Shim-pack IC-Al (4.6 mm i.d. x 100 mm) with the mixture of 1.8 mmol/L of phthalic acid and 1.35 mmol/L of Tri(hydroxymethyl) aminomethane as eluent. The flow rate of mobile phase is 1.0 mL/min. The injection volume was 20 microL. The column oven temperature was controlled at 40 degrees C. Hydroxyethyl sulfonate anion and SO(3)2- could not be separated at the above conditions, but they did not interfere the determination of HCOO-. The detection limits for HCOO-, SO(3)2-, NO3-, S2O(3)2-, HOC2H4S2O3-, Cl- and SO(4)2- were 1.0, 0.7, 1.4, 5.0, 0.7, 0.2 and 2.0 mg/L, respectively. The recoveries were between 96%-102%, and the relative standard deviations (RSD) were lower than 6.0% (n = 5) for all seven ions. This method is characterized by rapidity, sensitivity and simultaneous determination of several kinds of ions.

  13. Separation of proteins by cation-exchange sequential injection chromatography using a polymeric monolithic column.

    PubMed

    Masini, Jorge Cesar

    2016-02-01

    Since sequential injection chromatography (SIC) emerged in 2003, it has been used for separation of small molecules in diverse samples, but separations of high molar mass compounds such as proteins have not yet been described. In the present work a poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic column was prepared by free radical polymerization inside a 2.1-mm-i.d. activated fused silica-lined stainless steel tubing and modified with iminodiacetic acid (IDA). The column was prepared from a mixture of 24% GMA, 16% EDMA, 20% cyclohexanol, and 40% 1-dodecanol (all% as w/w) containing 1% of azobisisobutyronitrile (AIBN) (in relation to monomers). Polymerization was done at 60 °C for 24 h. The polymer was modified with 1.0 M IDA (in 2 M Na2CO3, pH 10.5) at 80 °C for 16 h. Separation of myoglobin, ribonuclease A, cytochrome C, and lysozyme was achieved at pH 7.0 (20 mM KH2PO4/K2HPO4) using a salt gradient (NaCl). Myoglobin was not retained, and the other proteins were separated by a gradient of NaCl created inside the holding coil (4 m of 0.8-mm-i.d. PTFE tubing) by sequential aspiration of 750 and 700 μL of 0.2 and 0.1 M NaCl, respectively. As the flow was reversed toward the column (5 μL s(-1)) the interdispersion of these solutions created a reproducible gradient which separated the proteins in 10 min, with the following order of retention: ribonuclease A < cytochrome C < lysozyme. The elution order was consistent with a cation-exchange mechanism as the retention increased with the isoelectric points.

  14. Polar organic phase liquid chromatography with packed capillary columns using a vancomycin chiral stationary phase

    PubMed

    Svensson; Donnecke; Karlsson; Karlsson; Vessman

    2000-08-01

    Vancomycin immobilized on silica served as the chiral stationary phase (CSP) in this investigation with polar organic solvents as the mobile phase in liquid chromatography (LC). It was shown that trace amounts of water were beneficial for improving peak shape and efficiency. To regulate the retention and selectivity an acid and/or base were added to the mobile phase where an excess of acid was shown to be preferential for enantioseparation. An unusual increase in selectivity with increasing temperature was shown for the acidic drug, thalidomide. Additionally, nonlinear van't Hoff plots were obtained for metoprolol enantiomers that showed increased retention with increasing temperature. Metoprolol also showed unusual behavior in the polar organic phase when water was added to resemble reversed-phase chromatography, with minimum retention observed at high water or high methanol concentrations. In both instances a high degree of electrostatic interaction between metoprolol and vancomycin was concluded. Metoprolol and ten of its analogs were examined on this CSP to evaluate the enantiorecognition process. A comparison in enantioselectivity for a number of acidic and basic drugs using this CSP was also carried out using the polar organic phase, reversed phase, and normal phase LC which were all compared to the results obtained in supercritical fluid chromatography (SFC). Polar organic phase LC offered a better separation of basic molecules while reversed phase LC was preferred for the resolution of acids. SFC showed the broadest enantioselectivity overall and normal phase LC indicated similar properties, as expected, to SFC but with lower column efficiency. Copyright 2000 Wiley-Liss, Inc. PMID:10897097

  15. Simplifying the synthesis of SIgA: combination of dIgA and rhSC using affinity chromatography

    PubMed Central

    Moldt, Brian; Saye-Francisco, Karen; Schultz, Niccole; Burton, Dennis R.; Hessell, Ann J.

    2013-01-01

    The mucosal epithelia together with adaptive immune responses, such as local production and secretion of dimeric and polymeric immunoglobulin A (IgA), are a crucial part of the first line of defense against invading pathogens. IgA is primarily secreted as SIgA and plays multiply roles in mucosal defense. The study of SIgA-mediated protection is an important area of research in mucosal immunity but an easy, fast and reproducible method to generate pathogen-specific SIgA in vitro has not been available. We report here a new method to produce SIgA by co-purification of dimeric IgA, containing J chain, and recombinant human SC expressed in CHO cells. We previously reported the generation, production and characterization of the human recombinant monoclonal antibody IgA2 b12. This antibody, derived from the variable regions of the neutralizing anti-HIV-1 mAb IgG1 b12, blocked viral attachment and uptake by epithelial cells in vitro. We used a cloned CHO cell line that expresses monomeric, dimeric and polymeric species of IgA2 b12 for large-scale production of dIgA2 b12. Subsequently, we generated a CHO cell line to express recombinant human secretory component (rhSC). Here, we combined dIgA2 b12 and CHO-expressed rhSC via column chromatography to produce SIgA2 b12 that remains fully intact upon elution with 0.1M Citric acid, pH 3.0. We have performed biochemical analysis of the synthesized SIgA to confirm the species is of the expected size and retains the functional properties previously described for IgA2 b12. We show that SIgA2 b12 binds to the HIV-1 gp120 glycoprotein with similar apparent affinity to that of monomeric and dimeric forms of IgA2 b12 and neutralizes HIV-1 isolates with similar potency. An average yield of 6 mg of SIgA2 b12 was achieved from the combination of 20 mg of purified dIgA2 b12 and 2 L of rhSC-containing CHO cell supernatant. We conclude that synthesized production of stable SIgA can be generated by co-purification. This process introduces a

  16. A protocol for the measurement of all the parameters of the mass transfer kinetics in columns used in liquid chromatography

    SciTech Connect

    Gritti, Fabrice; Guiochon, Georges A

    2010-01-01

    Band broadening in chromatography results from the combination of the dispersive effects that are associated with the different steps involved in the migration of compound bands along the column. These steps include longitudinal diffusion, trans-particle mass transfer, external film mass transfer, overall eddy diffusion, including trans-column, short-range inter-channel, trans-channel eddy diffusion, and the possible, additional mass transfer contributions arising from heat friction and the thermal heterogeneity of the column. We describe a series of experiments that provide the data needed to determine the coefficients of the contributions to band broadening of each one of these individual mass transfer steps. This specifically designed protocol can provide key information regarding the kinetic performance of columns used in liquid chromatography and explain why different columns behave so differently. The limitations, accuracy and precision of these methods are discussed. Further avenues of research that could improve the characterization of the mass transfer mechanisms in chromatographic columns, possibly contributing to the development of better columns, are suggested.

  17. Characterization of column packing materials in high-performance liquid chromatography by charge-detection quadrupole ion trap mass spectrometry.

    PubMed

    Xiong, Caiqiao; Zhou, Xiaoyu; Chen, Rui; Zhang, Yiming; Peng, Wen-Ping; Nie, Zongxiu; Chang, Huan-Cheng; Liu, Huwei; Chen, Yi

    2011-07-01

    This article reports an application of charge-detection quadrupole ion trap mass spectrometry (CD-ITMS) to characterize the column packing materials in high-performance liquid chromatography (HPLC). Both the mean mass and the mass distribution of the packing materials are obtained and used to calculate the specific surface area of unbonded silica, the carbon load of the bonded silica, and their particle size distributions. The obtained specific surface areas and carbon loads are consistent with those measured independently by nitrogen sorption and elemental analysis respectively, whereas the derived size distributions show better resolution than that measured by a laser particle size analyzer. Furthermore, we evaluate the uniformity of particle size, which is the key parameter for column efficiency of the liquid chromatography by analyzing the mass distribution of the packing materials at the top and bottom of the column. A broader mass distribution, which yields decreased column efficiency, is observed for the column top because of the excessive use of the column. Our results suggest that CD-ITMS can serve as an alternative means for the characterization of the packing materials in HPLC and is potentially useful for column quality control.

  18. Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry.

    PubMed

    Aryal, Uma K; Ross, Andrew R S

    2010-01-01

    Titanium dioxide metal oxide affinity chromatography (TiO(2)-MOAC) is widely regarded as being more selective than immobilized metal-ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO(2)-MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO(2)-MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non-phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5-dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO(2)-MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO(2)-MOAC showed higher specificity than immobilized gallium (Ga(3+)), immobilized iron (Fe(3+)), or zirconium dioxide (ZrO(2)) affinity chromatography for phosphopeptide enrichment. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), which was more efficient for smaller, singly phosphorylated peptides. PMID:20014058

  19. Using Artificial Soil and Dry-Column Flash Chromatography to Simulate Organic Substance Leaching Process: A Colorful Environmental Chemistry Experiment

    ERIC Educational Resources Information Center

    de Avellar, Isa G. J.; Cotta, Tais A. P. G.; Neder, Amarilis de V. Finageiv

    2012-01-01

    Soil is an important and complex environmental compartment and soil contamination contributes to the pollution of aquifers and other water basins. A simple and low-cost experiment is described in which the mobility of three organic compounds in an artificial soil is examined using dry-column flash chromatography. The compounds were applied on top…

  20. Affinity column for purification of the human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor

    SciTech Connect

    Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

    1987-05-01

    The TXA/sub 2//PGH/sub 2/ receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific /sup 3/H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA/sub 2//PGH/sub 2/ receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor.

  1. Optimal performance of single-column chromatography and simulated moving bed processes for the separation of optical isomers

    NASA Astrophysics Data System (ADS)

    Medi, Bijan; Kazi, Monzure-Khoda; Amanullah, Mohammad

    2013-06-01

    Chromatography has been established as the method of choice for the separation and purification of optically pure drugs which has a market size of about 250 billion USD. Single column chromatography (SCC) is commonly used in the development and testing phase of drug development while multi-column Simulated Moving Bed (SMB) chromatography is more suitable for large scale production due to its continuous nature. In this study, optimal performance of SCC and SMB processes for the separation of optical isomers under linear and overloaded separation conditions has been investigated. The performance indicators, namely productivity and desorbent requirement have been compared under geometric similarity for the separation of a mixture of guaifenesin, and Tröger's base enantiomers. SCC process has been analyzed under equilibrium assumption i.e., assuming infinite column efficiency, and zero dispersion, and its optimal performance parameters are compared with the optimal prediction of an SMB process by triangle theory. Simulation results obtained using actual experimental data indicate that SCC may compete with SMB in terms of productivity depending on the molecules to be separated. Besides, insights into the process performances in terms of degree of freedom and relationship between the optimal operating point and solubility limit of the optical isomers have been ascertained. This investigation enables appropriate selection of single or multi-column chromatographic processes based on column packing properties and isotherm parameters.

  2. Trend analysis of performance parameters of pre-packed columns for protein chromatography over a time span of ten years.

    PubMed

    Scharl, Theresa; Jungreuthmayer, Christian; Dürauer, Astrid; Schweiger, Susanne; Schröder, Tim; Jungbauer, Alois

    2016-09-23

    Pre-packed small scale chromatography columns are increasingly used for process development, for determination of design space in bioprocess development, and for post-licence process verifications. The packing quality of 30,000 pre-packed columns delivered to customers over a period 10 years has been analyzed by advanced statistical tools. First, the data were extracted and checked for inconsistencies, and then were tabulated and made ready for statistical processing using the programming language Perl (https://www.perl.org/) and the statistical computing environment R (https://www.r-project.org/). Reduced HETP and asymmetry were plotted over time to obtain a trend of packing quality over 10 years. The obtained data were used as a visualized coefficient of variation analysis (VCVA), a process that has often been applied in other industries such as semiconductor manufacturing. A typical fluctuation of reduced HETP was seen. A Tsunami effect in manufacturing, the effect of propagation of manufacturing deviations leading to out-of-specification products, was not observed with these pre-packed columns. Principal component analysis (PCA) showed that all packing materials cluster. Our data analysis showed that the current commercially available chromatography media used for biopharmaceutical manufacturing can be reproducibly and uniformly packed in polymer-based chromatography columns, which are designed for ready-to-use purposes. Although the number of packed columns has quadrupled over one decade the packing quality has remained stable.

  3. Trend analysis of performance parameters of pre-packed columns for protein chromatography over a time span of ten years.

    PubMed

    Scharl, Theresa; Jungreuthmayer, Christian; Dürauer, Astrid; Schweiger, Susanne; Schröder, Tim; Jungbauer, Alois

    2016-09-23

    Pre-packed small scale chromatography columns are increasingly used for process development, for determination of design space in bioprocess development, and for post-licence process verifications. The packing quality of 30,000 pre-packed columns delivered to customers over a period 10 years has been analyzed by advanced statistical tools. First, the data were extracted and checked for inconsistencies, and then were tabulated and made ready for statistical processing using the programming language Perl (https://www.perl.org/) and the statistical computing environment R (https://www.r-project.org/). Reduced HETP and asymmetry were plotted over time to obtain a trend of packing quality over 10 years. The obtained data were used as a visualized coefficient of variation analysis (VCVA), a process that has often been applied in other industries such as semiconductor manufacturing. A typical fluctuation of reduced HETP was seen. A Tsunami effect in manufacturing, the effect of propagation of manufacturing deviations leading to out-of-specification products, was not observed with these pre-packed columns. Principal component analysis (PCA) showed that all packing materials cluster. Our data analysis showed that the current commercially available chromatography media used for biopharmaceutical manufacturing can be reproducibly and uniformly packed in polymer-based chromatography columns, which are designed for ready-to-use purposes. Although the number of packed columns has quadrupled over one decade the packing quality has remained stable. PMID:27575920

  4. Coupled achiral/chiral column techniques in subcritical fluid chromatography for the separation of chiral and nonchiral compounds.

    PubMed

    Phinney, K W; Sander, L C; Wise, S A

    1998-06-01

    A multicolumn approach was developed to address the limited achiral selectivity of chiral stationary phases. Groups of structurally related compounds, including beta-blockers and 1,4-benzodiazepines, were separated using coupled achiral/chiral stationary phases under subcritical fluid conditions. The achiral selectivity of amino and cyano stationary phases was used to modify the resolution of compounds on a Chiralcel OD chiral stationary phase by combining the achiral and chiral columns in series. In the case of the benzodiazepines, separation of achiral compounds was performed concurrently with the enantioseparation of chiral molecules. The separation of components of a multidrug cough and cold medication was also demonstrated on a cyano column coupled with a Chiralpak AD chiral stationary phase. The use of modified carbon dioxide eluents eliminated the mobile phase incompatibility problems associated with column coupling in liquid chromatography and incorporated the high efficiency of sub- and supercritical fluid chromatography.

  5. A Proteomics Platform Combining Depletion, Multi-lectin Affinity Chromatography (M-LAC) and Isoelectric Focusing to Study the Breast Cancer Proteome

    PubMed Central

    Zeng, Zhi; Hincapie, Marina; Pitteri, Sharon J.; Hanash, Samir; Schalkwijk, Joost; Hogan, Jason M.; Wang, Hon; Hancock, William S.

    2011-01-01

    The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as simultaneously detecting glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation and LC-MS analysis has been applied to discover breast cancer associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies. PMID:21513341

  6. Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry.

    PubMed

    Zhan, Xianquan; Desiderio, Dominic M

    2006-07-15

    The aim of this study was to characterize endogenous nitroproteins, and those proteins that interact with nitroproteins, in a human pituitary nonfunctional adenoma so as to clarify the role of protein nitration in adenomas. A nitrotyrosine affinity column (NTAC) was used to preferentially enrich and isolate endogenous nitroproteins and nitroprotein-protein complexes from a tissue homogenate that was prepared from a human pituitary nonfunctional pituitary adenoma. The preferentially enriched endogenous nitroproteins and nitroprotein-protein complexes were subjected to trypsin digestion, desalination, and tandem mass spectrometry analysis. Nine nitroproteins (Rho-GTPase-activing protein 5, leukocyte immunoglobulin-like receptor subfamily A member 4 precursor, zinc finger protein 432, cAMP-dependent protein kinase type I-beta regulatory subunit, sphingosine-1-phosphate lyase 1, centaurin beta 1, proteasome subunit alpha type 2, interleukin 1 family member 6, and rhophilin 2) and three proteins (interleukin 1 receptor-associated kinase-like 2, glutamate receptor-interacting protein 2, and ubiquitin) that interacted with nitroproteins were discovered. The nitration site of each nitroprotein was located onto the functional domain where nitration occurred, and each nitroprotein was related to a corresponding functional system. Those data indicate that protein nitration might be an important molecular event in the formation of a human pituitary nonfunctional adenoma.

  7. Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns.

    PubMed

    Bi, Cong; Zheng, Xiwei; Hage, David S

    2016-02-01

    In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. PMID:26797422

  8. Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography*

    PubMed Central

    Hoehenwarter, Wolfgang; Thomas, Martin; Nukarinen, Ella; Egelhofer, Volker; Röhrig, Horst; Weckwerth, Wolfram; Conrath, Uwe; Beckers, Gerold J. M.

    2013-01-01

    Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment. PMID:23172892

  9. Evaluation of reversible interconversion in comprehensive two-dimensional gas chromatography using enantioselective columns in first and second dimensions.

    PubMed

    Kröger, Sabrina; Wong, Yong Foo; Chin, Sung-Tong; Grant, Jacob; Lupton, David; Marriott, Philip J

    2015-07-24

    The reversible molecular interconversion behaviour of a synthesised oxime (2-phenylpropanaldehyde oxime; (C6H5)CH(CH3)CHN(OH)) was investigated by both, single dimensional gas chromatography (1D GC) and comprehensive two-dimensional gas chromatography (GC×GC). Previous studies on small molecular weight oximes were extended to this larger aromatic oxime (molar mass 149.19gmol(-1)) with interest in the extent of interconversion, enantioselective resolution, and retention time. On a polyethylene glycol (PEG; wax-type) column, a characteristic interconversion zone between two antipodes of E and Z isomers was formed by molecules which have undergone isomerisation on the column (E⇌Z). The extent of interconversion was investigated by varying chromatographic conditions (oven temperature and carrier flow rate) to understand the nature of the behaviour observed. The extent of interconversion was negligible in both enantioselective and methyl-phenylpolysiloxane phase-columns, correlating with the low polarity of the stationary phase. In order to obtain isomerisation along with enantio-resolution, a wax-type and an enantioselective column were coupled in either enantioselective-wax or wax-enantioselective order. The most appropriate column arrangement was selected for study by using a GC×GC experiment with either a wax-phase or phenyl-methylpolysiloxane phase as (2)D column. In addition to evaluation of these fast elution columns, a long narrow-bore enantioselective column (10m) was introduced as (2)D, providing an enantioselective-PEG (coupled-column ensemble: (1)D1+(1)D2)×enantioselective ((2)D) column combination. In this instance, the (1)D1 enantioselective column provides enantiomeric separation of the corresponding enantiomers ((R) and (S)) of (E)- and (Z)-2-phenylpropanaldehyde oxime, followed by E/Z isomerisation in the coupled (1)D2 PEG (reactor) column. The resulting chromatographic interconversion region was modulated and separated into either E/Z isomers

  10. Analysis of drugs in plasma samples from schizophrenic patients by column-switching liquid chromatography-tandem mass spectrometry with organic-inorganic hybrid cyanopropyl monolithic column.

    PubMed

    Domingues, Diego Soares; Souza, Israel Donizeti de; Queiroz, Maria Eugênia Costa

    2015-07-01

    This study reports on the development of a rapid, selective, and sensitive column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyze sixteen drugs (antidepressants, anticonvulsants, anxiolytics, and antipsychotics) in plasma samples from schizophrenic patients. The developed organic-inorganic hybrid monolithic column with cyanopropyl groups was used for the first dimension of the column-switching arrangement. This arrangement enabled online pre-concentration of the drugs (monolithic column) and their subsequent analytical separation on an XSelect SCH C18 column. The drugs were detected on a triple quadrupole tandem mass spectrometer (multiple reactions monitoring mode) with an electrospray ionization source in the positive ion mode. The developed method afforded adequate linearity for the sixteen target drugs; the coefficients of determination (R(2)) lay above 0.9932, the interassay precision had coefficients of variation lower than 6.5%, and the relative standard error values of the accuracy ranged from -14.0 to 11.8%. The lower limits of quantification in plasma samples ranged from 63 to 1250pgmL(-1). The developed method successfully analyzed the target drugs in plasma samples from schizophrenic patients for therapeutic drug monitoring (TDM).

  11. Screening of Potential Xanthine Oxidase Inhibitors in Gnaphalium hypoleucum DC. by Immobilized Metal Affinity Chromatography and Ultrafiltration-Ultra Performance Liquid Chromatography-Mass Spectrometry.

    PubMed

    Zhang, Hong-Jian; Hu, Yi-Juan; Xu, Pan; Liang, Wei-Qing; Zhou, Jie; Liu, Pei-Gang; Cheng, Lin; Pu, Jin-Bao

    2016-01-01

    In this study, a new method based on immobilized metal affinity chromatography (IMAC) combined with ultrafiltration-ultra performance liquid chromatography-mass spectrometry (UF-UPLC-MS) was developed for discovering ligands for xanthine oxidase (XO) in Gnaphalium hypoleucum DC., a folk medicine used in China for the treatment of gout. By IMAC, the high flavonoid content of G. hypoleucum could be determined rapidly and efficiently. UF-UPLC-MS was used to select the bound xanthine oxidase ligands in the mixture and identify them. Finally, two flavonoids, luteolin-4'-O-glucoside and luteolin, were successfully screened and identified as the candidate XO inhibitors of G. hypoleucum. They were evaluated in vitro for XO inhibitory activity and their interaction mechanism was studied coupled with molecular simulations. The results were in favor of the hypothesis that the flavonoids of G. hypoleucum might be the active content for gout treatment by inhibiting XO. PMID:27649136

  12. 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. PMID:23978749

  13. Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

    PubMed Central

    Locatelli-Hoops, Silvia C.; Yeliseev, Alexei A.

    2016-01-01

    Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor. PMID:24943318

  14. Comparison of hypercrosslinked polystyrene columns for the separation of nitrogen group-types in petroleum using High Performance Liquid Chromatography.

    PubMed

    Oro, Nicole E; Lucy, Charles A

    2010-10-01

    High performance liquid chromatography in a quasi-normal phase mode (QNP) is used to separate the nitrogen group-types (pyrrole and pyridine) that are found in petroleum. A new type of stationary phase, hypercrosslinked polystyrene, is used to achieve this separation. Three different hypercrosslinked polystyrene stationary phases are compared under quasi-normal phase mode; a commercial 5-HGN packing, and two hypercrosslinked phases on silica particles. The utility of the columns for petroleum-based separations was explored with the use of 21 analytical standards. Partial elucidation of adsorption retention mechanisms for the columns are shown, as well as a comparison of retention characteristics for the three columns. The silica particle column derived with toluene (HC-Tol) was found to have the best selectivity for nitrogen group-types and polycyclic aromatic hydrocarbons (PAHs), attaining a separation under gradient conditions in less than 30 min.

  15. A reduced order model for the study of asymmetries in linear gas chromatography for homogeneous tubular columns.

    SciTech Connect

    Whiting, Joshua J.; Romero, Louis Anthony; Parks, Michael L.

    2005-08-01

    In gas chromatography, a chemical sample separates into its constituent components as it travels along a long thin column. As the component chemicals exit the column they are detected and identified, allowing the chemical makeup of the sample to be determined. For correct identification of the component chemicals, the distribution of the concentration of each chemical along the length of the column must be nearly symmetric. The prediction and control of asymmetries in gas chromatography has been an active research area since the advent of the technique. In this paper, we develop from first principles a general model for isothermal linear chromatography. We use this model to develop closed-form expressions for terms related to the first, second, and third moments of the distribution of the concentration, which determines the velocity, diffusion rate, and asymmetry of the distribution. We show that for all practical experimental situations, only fronting peaks are predicted by this model, suggesting that a nonlinear chromatography model is required to predict tailing peaks. For situations where asymmetries arise, we analyze the rate at which the concentration distribution returns to a normal distribution. Numerical examples are also provided.

  16. Purification of sulforaphane from Brassica oleracea seed meal using low-pressure column chromatography.

    PubMed

    Liang, Hao; Yuan, Qipeng; Xiao, Qian

    2005-12-15

    Sulforaphane is an isothiocyanate that is present naturally in widely consumed Brassica oleracea vegetables and has been shown to block the formation of tumors. The contents of sulforaphane in five groups of B. oleracea seeds (broccoli, Brussels sprouts, cabbage, cauliflower and kale) were determined by RP-HPLC using linear gradient of acetonitrile in water. A new low-cost method to isolate and purify natural sulforaphane from B. oleracea seed meal was described in this work. Crude sulforaphane was first separated from B. oleracea seed meal by using immiscible solvent extraction with ethyl acetate, 10% ethanol and hexane, and the crude sulforaphane was used as raw materials to prepare high purity sulforaphane by low-pressure column chromatography of silica gel (200-300 mesh) with different eluents and elution modes. Compared with these different elution methods, the gradient elution was preferable to the isocratic elution for reducing the elution time and the eluent consumption and increasing the purity of sulforaphane product. The purity and recovery of sulforaphane were more than 90% in gradient elution.

  17. High purity isolation and quantification of semiconducting carbon nanotubes via column chromatography.

    PubMed

    Tulevski, George S; Franklin, Aaron D; Afzali, Ali

    2013-04-23

    The isolation of semiconducting carbon nanotubes (CNTs) to ultrahigh (ppb) purity is a prerequisite for their integration into high-performance electronic devices. Here, a method employing column chromatography is used to isolate semiconducting nanotubes to 99.9% purity. The study finds that by modifying the solution preparation step, both the metallic and semiconducting fraction are resolved and elute using a single surfactant system, allowing for multiple iterations. Iterative processing enables a far more rapid path to achieving the level of purities needed for high performance computing. After a single iteration, the metallic peak in the absorption spectra is completely attenuated. Although absorption spectroscopy is typically used to characterize CNT purity, it is found to be insufficient in quantifying solutions of high purity (>98 to 99%) due to low signal-to-noise in the metallic region of ultrahigh purity solutions. Therefore, a high throughput electrical testing method was developed to quantify the degree of separation by characterizing ∼4000 field-effect transistors fabricated from the separated nanotubes after multiple iterations of the process. The separation and characterization methods described here provide a path to produce the ultrahigh purity semiconducting CNT solutions needed for high performance electronics. PMID:23484490

  18. Studies on the Performance of Different Coiled Column Configurations for Compact Type-I Counter-current Chromatography

    PubMed Central

    Yang, Yi; Gu, Dongyu; Aisa, Haji Akber; Ito, Yoichiro

    2011-01-01

    Three types of novel coiled column configurations, i.e., a triangular coiled column and elliptical coiled columns I and II, were designed for type-I countercurrent chromatography and their performances were evaluated with two solvent systems each with suitable test samples. Three DNP-amino acids (DNP-DL-glu, DNP-β-ala and DNP-L-ala) were separated with a moderately hydrophobic two-phase solvent system composed of hexane-ethyl acetate-metanol-0.1M hydrochloric acid (1:1:1:1, v/v), while two dipeptides (tryptophyl tyrosine and valyl-tyrosine) were separated with a polar solvent system composed of 1-butanol-acetic acid-water (4.75:0.25:5, v/v). The overall results indicated that the performance of compact type-I counter-current chromatography was improved by elliptical coiled column II which was mounted with its maximum coil diameter perpendicular to the surface of the column holder. Hydrodynamic effects involved in these separations were discussed. PMID:21491597

  19. Greener liquid chromatography using a guard column with micellar mobile phase for separation of some pharmaceuticals and determination of parabens.

    PubMed

    Youngvises, Napaporn; Chaida, Thanatcha; Khonyoung, Supada; Kuppithayanant, Nattawan; Tiyapongpattana, Warawut; Itharat, Arunporn; Jakmunee, Jaroon

    2013-03-15

    In this research, a greener chromatography employing a short column, Zorbax SB C18 cartridge (12.5 × 4.6 mm, 5 μm) commonly used as a guard column in a reverse phase high performance liquid chromatography (RP-HPLC), was utilized as the analytical column in conjunction with a more eco-friendly micellar mobile phase of sodium dodecyl sulfate (SDS) for separation tertiary mixtures of local anesthetics and antihistamines; and binary mixture of colds drugs; and quaternary mixture of some parabens with different separation conditions. The chromatographic behavior of these analytes was studied to demonstrate separation efficiency of this guard column in a micellar mobile phase. Moreover, this column and SDS mobile phase was exploited for determination of parabens in 64 samples of cosmetic product, both those that were produced locally in the community and those that were commercially manufactured. Linear calibration graphs of the parabens as detected at 254 nm were obtained in the range of 1-100 μmol L(-1) with R(2)>0.9990. Percentage recoveries were 92.4-109.2 with %RSD<3, and the limit of detection and quantitation were 0.04-0.10 and 0.20-0.80 μmol L(-1), respectively. This analytical system is not only greener but also faster and employing simpler sample preparation than a conventional liquid chromatographic system. PMID:23598137

  20. Greener liquid chromatography using a guard column with micellar mobile phase for separation of some pharmaceuticals and determination of parabens.

    PubMed

    Youngvises, Napaporn; Chaida, Thanatcha; Khonyoung, Supada; Kuppithayanant, Nattawan; Tiyapongpattana, Warawut; Itharat, Arunporn; Jakmunee, Jaroon

    2013-03-15

    In this research, a greener chromatography employing a short column, Zorbax SB C18 cartridge (12.5 × 4.6 mm, 5 μm) commonly used as a guard column in a reverse phase high performance liquid chromatography (RP-HPLC), was utilized as the analytical column in conjunction with a more eco-friendly micellar mobile phase of sodium dodecyl sulfate (SDS) for separation tertiary mixtures of local anesthetics and antihistamines; and binary mixture of colds drugs; and quaternary mixture of some parabens with different separation conditions. The chromatographic behavior of these analytes was studied to demonstrate separation efficiency of this guard column in a micellar mobile phase. Moreover, this column and SDS mobile phase was exploited for determination of parabens in 64 samples of cosmetic product, both those that were produced locally in the community and those that were commercially manufactured. Linear calibration graphs of the parabens as detected at 254 nm were obtained in the range of 1-100 μmol L(-1) with R(2)>0.9990. Percentage recoveries were 92.4-109.2 with %RSD<3, and the limit of detection and quantitation were 0.04-0.10 and 0.20-0.80 μmol L(-1), respectively. This analytical system is not only greener but also faster and employing simpler sample preparation than a conventional liquid chromatographic system.

  1. High-performance liquid chromatography separation of unsaturated organic compounds by a monolithic silica column embedded with silver nanoparticles.

    PubMed

    Zhu, Yang; Morisato, Kei; Hasegawa, George; Moitra, Nirmalya; Kiyomura, Tsutomu; Kurata, Hiroki; Kanamori, Kazuyoshi; Nakanishi, Kazuki

    2015-08-01

    The optimization of a porous structure to ensure good separation performances is always a significant issue in high-performance liquid chromatography column design. Recently we reported the homogeneous embedment of Ag nanoparticles in periodic mesoporous silica monolith and the application of such Ag nanoparticles embedded silica monolith for the high-performance liquid chromatography separation of polyaromatic hydrocarbons. However, the separation performance remains to be improved and the retention mechanism as compared with the Ag ion high-performance liquid chromatography technique still needs to be clarified. In this research, Ag nanoparticles were introduced into a macro/mesoporous silica monolith with optimized pore parameters for high-performance liquid chromatography separations. Baseline separation of benzene, naphthalene, anthracene, and pyrene was achieved with the theoretical plate number for analyte naphthalene as 36,000 m(-1). Its separation function was further extended to cis/trans isomers of aromatic compounds where cis/trans stilbenes were chosen as a benchmark. Good separation of cis/trans-stilbene with separation factor as 7 and theoretical plate number as 76,000 m(-1) for cis-stilbene was obtained. The trans isomer, however, is retained more strongly, which contradicts the long- established retention rule of Ag ion chromatography. Such behavior of Ag nanoparticles embedded in a silica column can be attributed to the differences in the molecular geometric configuration of cis/trans stilbenes.

  2. Effect of high-temperature on high-performance liquid chromatography column stability and performance under temperature-programmed conditions.

    PubMed

    Marin, Stephanie J; Jones, Brian A; Felix, W Dale; Clark, Jody

    2004-03-19

    Six commercially available analytical (4.1 or 4.6 mm i.d.) columns were evaluated under temperature-programmed high-temperature liquid chromatography (HTLC) conditions to access their stability and performance at extreme temperatures. Seven components consisting of acidic, basic and neutral compounds were analyzed under temperature-programmed conditions and solvent gradient conditions using three different mobile phase compositions (acidic, basic and neutral). Each column was checked with a two-component test mix at various stages of the evaluation to look for signs of stationary phase collapse. Three zirconia based stationary phases studied exhibited column bleed under temperature-programmed conditions. The other three columns, a polydentate silica column, a polystyrene-divinylbenzene (PS-DVB) polymeric column, and a graphitic carbon column performed well with no evidence of stationary phase degradation. The R.S.D. for the retention times and efficiencies were less than 10% for most conditions, and not more than 15% during the course of the evaluation for each column. The polydentate silica stationary phase was temperature programmed to 100 degrees C, the PS-DVB stationary phase was temperature programmed up to 150 degrees C, and the graphitic carbon column was used with temperature programming up to 200 degrees C. Comparable peak capacities and similar retention behaviors were observed under solvent gradient and temperature-programmed conditions. Temperature programming with dynamic mobile phase preheating can replace solvent gradient analysis without a loss of peak capacity when used with 4.1 or 4.6 mm columns. PMID:15043277

  3. Half-width plots, a simple tool to predict peak shape, reveal column kinetics and characterise chromatographic columns in liquid chromatography: state of the art and new results.

    PubMed

    Baeza-Baeza, J J; Ruiz-Ángel, M J; García-Álvarez-Coque, M C; Carda-Broch, S

    2013-11-01

    Peak profiles in chromatography are characterised by their height, position, width and asymmetry; the two latter depend on the values of the left and right peak half-widths. Simple correlations have been found between the peak half-widths and the retention times. The representation of such correlations has been called half-width plots. For isocratic elution, the plots are parabolic, although often, the parabolas can be approximated to straight-lines. The plots can be obtained with the half-widths/retention time data for a set of solutes experiencing the same kinetics, eluted with a mobile phase at fixed or varying composition. When the analysed solutes experience different resistance to mass transfer, the plots will be solute dependent, and should be obtained with the data for each solute eluted with mobile phases at varying composition. The half-width plots approach is a simple tool that facilitates the prediction of peak shape (width and asymmetry) with optimisation purposes, reveal the interaction kinetics of solutes in different columns, and characterise chromatographic columns. This work shows half-width plots for different situations in isocratic elution, including the use of different flows, the effect of temperature, the modification of the stationary phase surface by an additive, the existence of specific interactions within the column, and the comparison of columns. The adaptation to gradient elution is also described. Previous knowledge on half-width plots is structured and analysed, to which new results are added.

  4. Interference between eplerenone and digoxin in fluorescence polarization immunoassay, microparticle enzyme immunoassay, and affinity column-mediated immunoassay.

    PubMed

    Yamada, Tomoyuki; Suzuki, Kaoru; Iguchi, Ken; Kanada, Yasutaka; Kato, Ryuji; Ijiri, Yoshio; Nishihara, Masami; Murakami, Sumiko; Hayashi, Tetsuya; Tamai, Hiroshi; Tanaka, Kazuhiko

    2010-12-01

    Digitalis-like immunoreactive substances have crossreactivity with antidigoxin antibodies and the interference between digoxin and spironolactone/canrenone has been reported. The structure of eplerenone is similar to that of spironolactone/canrenone. Therefore, we hypothesized that eplerenone might also interfere with the measurement of digoxin by immunoassay. We performed three types of assays (fluorescence polarization immunoassay [FPIA], microparticle enzyme immunoassay [MEIA], and affinity column-mediated immunoassay [ACMIA]) to determine crossreactions between eplerenone and antidigoxin antibodies. Furthermore, we used FPIA, MEIA, and ACMIA to measure the apparent digoxin concentration in mixed solutions of eplerenone (1-100 μg/mL) and digoxin (1-3 ng/mL). In the crossreaction tests, eplerenone was detected as digoxin by FPIA and ACMIA. By FPIA, a known concentration of 1 μg/mL of eplerenone was measured as 0.33 ± 0.11 ng/mL of digoxin (crossreaction rate, 0.03%). By ACMIA, a known concentration of 10 μg/mL of eplerenone was measured as 0.13 ± 0.05 ng/mL of digoxin (crossreaction rate, 0.001%). No crossreaction between eplerenone and digoxin was determined by MEIA. In the interference of eplerenone coadministered with digoxin, the apparent concentration of digoxin was increased in FPIA, but decreased in MEIA and ACMIA. The results suggest that eplerenone crossreacts with antidigoxin antibodies in FPIA, MEIA, and ACMIA, but that the interference of eplerenone might be smaller than that of spironolactone/canrenone. PMID:20625353

  5. Oxidation of element 102, nobelium, with flow electrolytic column chromatography on an atom-at-a-time scale.

    PubMed

    Toyoshima, Atsushi; Kasamatsu, Yoshitaka; Tsukada, Kazuaki; Asai, Masato; Kitatsuji, Yoshihiro; Ishii, Yasuo; Toume, Hayato; Nishinaka, Ichiro; Haba, Hiromitsu; Ooe, Kazuhiro; Sato, Wataru; Shinohara, Atsushi; Akiyama, Kazuhiko; Nagame, Yuichiro

    2009-07-01

    We report here on the successful oxidation of element 102, nobelium (No), on an atom-at-a-time scale in 0.1 M alpha-hydroxyisobutyric acid (alpha-HIB) solution using a newly developed technique, flow electrolytic column chromatography. It is found that the most stable ion, No(2+), is oxidized to No(3+) within 3 min and that the oxidized No complex with alpha-HIB holds the trivalent state in the column above an applied potential of 1.0 V.

  6. Oxidation of element 102, nobelium, with flow electrolytic column chromatography on an atom-at-a-time scale.

    PubMed

    Toyoshima, Atsushi; Kasamatsu, Yoshitaka; Tsukada, Kazuaki; Asai, Masato; Kitatsuji, Yoshihiro; Ishii, Yasuo; Toume, Hayato; Nishinaka, Ichiro; Haba, Hiromitsu; Ooe, Kazuhiro; Sato, Wataru; Shinohara, Atsushi; Akiyama, Kazuhiko; Nagame, Yuichiro

    2009-07-01

    We report here on the successful oxidation of element 102, nobelium (No), on an atom-at-a-time scale in 0.1 M alpha-hydroxyisobutyric acid (alpha-HIB) solution using a newly developed technique, flow electrolytic column chromatography. It is found that the most stable ion, No(2+), is oxidized to No(3+) within 3 min and that the oxidized No complex with alpha-HIB holds the trivalent state in the column above an applied potential of 1.0 V. PMID:19514720

  7. Determination of free and total sulfate and phosphate in glycosaminoglycans by column-switching high-performance size-exclusion and ion chromatography and single-column ion chromatography.

    PubMed

    Ruiz-Calero, V; Puignou, L; Diez, M; Galceran, M T

    2001-02-01

    Analytical procedures for the determination of free and total sulfate and phosphate in glycosaminoglycans by high-performance liquid chromatography were studied. A column-switching method coupling high-performance size-exclusion chromatography (HPSEC) and ion chromatography (IC) is proposed for the determination of free anions. Good run-to-run and day-to-day precision values (RSD) of < 4.7% were obtained for both anions. Total anion contents were determined after wet acid hydrolysis with nitric acid-hydrogen peroxide (5 + 1) by single-column IC and ICP-AES elemental analysis in order to validate the results. Recoveries ranging from 94.6 to 99.0% for sulfate and from 80.8 to 94.0% for phosphate were obtained. Both HPSEC-IC and single-column IC methods were applied to the analysis of a low molecular mass heparin, a non-fractionated heparin and a chondroitin 4-sulfate. From the free and total sulfate determinations, the content of linked sulfur was calculated and ranged from 5.1 to 12.2% m/m. PMID:11235098

  8. Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching

    SciTech Connect

    Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.; Belchik, Sara M.; Shi, Liang; Monroe, Matthew E.; Smith, Richard D.; Lipton, Mary S.

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry-(LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed, or if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, a bacterial decaheme cytochrome, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded three- to six-fold more confident peptide-spectrum matches to heme-c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for four of the ten expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering nine out of ten sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 10-4 was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  9. Detection and identification of heme c-modified peptides by histidine affinity chromatography, high-performance liquid chromatography-mass spectrometry, and database searching.

    PubMed

    Merkley, Eric D; Anderson, Brian J; Park, Jea; Belchik, Sara M; Shi, Liang; Monroe, Matthew E; Smith, Richard D; Lipton, Mary S

    2012-12-01

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, two bacterial decaheme cytochromes, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded 3- to 6-fold more confident peptide-spectrum matches to heme c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for 4 of the 10 expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 1×10(-4) was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c. PMID:23082897

  10. A Highly Selective Hsp90 Affinity Chromatography Resin with a Cleavable Linker

    PubMed Central

    Hughes, Philip F; Barrott, Jared J; Carlson, David A; Loiselle, David R; Speer, Brittany L; Bodoor, Khaldon; Rund, Lauretta A; Haystead, Timothy A J

    2012-01-01

    Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media. PMID:22520629

  11. Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

    PubMed Central

    Brinkworth, R I; Masters, C J; Winzor, D J

    1975-01-01

    Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase. PMID:175784

  12. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

    PubMed Central

    Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E.; Yates, John R.

    2011-01-01

    In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases. PMID:21936497

  13. Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron-Transfer Dissociation Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Brock, Jonathan W.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John; Smith, Richard D.; Metz, Thomas O.

    2007-06-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resulted in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.

  14. Study on the Alkaloids in Tibetan Medicine Aconitum pendulum Busch by HPLC-MSn Combined with Column Chromatography.

    PubMed

    Wang, Beibei; Dong, Jie; Ji, Jiaojiao; Yuan, Jiang; Wang, Jiali; Wu, Jiarui; Tan, Peng; Liu, Yonggang

    2016-01-01

    A rapid, convenient and effective identification method of alkaloids was established and an attempt on isolating and analyzing the alkaloids in Aconitum pendulum Busch was conducted successfully. In this article, four high-content components including deoxyaconitine, benzoylaconine, aconine and neoline were isolated by using column chromatography. HPLC-MS(n)was employed to deduce the regulations of fragmentation of diterpenoid alkaloids which displayed a characteristic behavior of loss of CO(28u), CH3COOH(60u), CH3OH(32u), H2O(18u) and C6H5COOH(122u). Then, according to fragmentation regulation of mass spectrometry, 42 alkaloids were found inA. pendulum Among them, 38 compounds were identified and 29 alkaloids were reported for the first time for this herb. Therefore, this means that HPLC-MS(n)combined with column chromatography could work as an effective and reliable tool for rapid identification of the chemical components of herbal medicine.

  15. Study on the Alkaloids in Tibetan Medicine Aconitum pendulum Busch by HPLC-MSn Combined with Column Chromatography.

    PubMed

    Wang, Beibei; Dong, Jie; Ji, Jiaojiao; Yuan, Jiang; Wang, Jiali; Wu, Jiarui; Tan, Peng; Liu, Yonggang

    2016-01-01

    A rapid, convenient and effective identification method of alkaloids was established and an attempt on isolating and analyzing the alkaloids in Aconitum pendulum Busch was conducted successfully. In this article, four high-content components including deoxyaconitine, benzoylaconine, aconine and neoline were isolated by using column chromatography. HPLC-MS(n)was employed to deduce the regulations of fragmentation of diterpenoid alkaloids which displayed a characteristic behavior of loss of CO(28u), CH3COOH(60u), CH3OH(32u), H2O(18u) and C6H5COOH(122u). Then, according to fragmentation regulation of mass spectrometry, 42 alkaloids were found inA. pendulum Among them, 38 compounds were identified and 29 alkaloids were reported for the first time for this herb. Therefore, this means that HPLC-MS(n)combined with column chromatography could work as an effective and reliable tool for rapid identification of the chemical components of herbal medicine. PMID:26896350

  16. Determination of dissolved bromate in drinking water by ion chromatography and post column reaction: interlaboratory study.

    PubMed

    Cordeiro, Fernando; Robouch, Piotr; de la Calle, Maria Beatriz; Emteborg, Håkan; Charoud-Got, Jean; Schmitz, Franz

    2011-01-01

    A collaborative study, International Evaluation Measurement Programme-25a, was conducted in accordance with international protocols to determine the performance characteristics of an analytical method for the determination of dissolved bromate in drinking water. The method should fulfill the analytical requirements of Council Directive 98/83/EC (referred to in this work as the Drinking Water Directive; DWD). The new draft standard method under investigation is based on ion chromatography followed by post-column reaction and UV detection. The collaborating laboratories used the Draft International Organization for Standardization (ISO)/Draft International Standard (DIS) 11206 document. The existing standard method (ISO 15061:2001) is based on ion chromatography using suppressed conductivity detection, in which a preconcentration step may be required for the determination of bromate concentrations as low as 3 to 5 microg/L. The new method includes a dilution step that reduces the matrix effects, thus allowing the determination of bromate concentrations down to 0.5 microg/L. Furthermore, the method aims to minimize any potential interference of chlorite ions. The collaborative study investigated different types of drinking water, such as soft, hard, and mineral water. Other types of water, such as raw water (untreated), swimming pool water, a blank (named river water), and a bromate standard solution, were included as test samples. All test matrixes except the swimming pool water were spiked with high-purity potassium bromate to obtain bromate concentrations ranging from 1.67 to 10.0 microg/L. Swimming pool water was not spiked, as this water was incurred with bromate. Test samples were dispatched to 17 laboratories from nine different countries. Sixteen participants reported results. The repeatability RSD (RSD(r)) ranged from 1.2 to 4.1%, while the reproducibility RSD (RSDR) ranged from 2.3 to 5.9%. These precision characteristics compare favorably with those of ISO

  17. Preparative purification and desalting of bases and nucleosides labeled with tritium by column chromatography on sephadex G-10

    SciTech Connect

    Yalovleva, L.A.; Kaminskii, Y.L.; Kozyreva, O.I.; Nagorskii, A.I.; Patokina, N.A.; Sosnova, L.P.

    1986-03-01

    The authors demonstrate the application of column chromatography on Sephadex G-10 and elution with water for the isolation of tritium labeled components of nucleic acids from reaction mixtures after catalytic dehalogenation or enzymic desoxyribosylation and simultaneous removal from inorganic salts. Distribution constants of 16 bases and nucleosides on elution with water were determined. Comparison of the sorbents with Sephadex G-20 disclosed the undoubted advantages of the latter in processes of desalting and separation of mixtures of bases and nucleosides.

  18. Analysis of phytochelatins in plant matrices by pre-column derivatization, high-performance liquid chromatography and fluorescence-detection.

    PubMed

    Döring, S; Korhammer, S; Oetken, M; Markert, B

    2000-02-01

    A sensitive method for the determination of phytochelatins in plant matrices by pre-column derivatization with monobromobimane (mBrB) and high-performance liquid chromatography (HPLC) on reversed phases and fluorescence-detection has been developed and applied to cucumber sprouts (Cucumis sativus) treated with cadmium and to the water moss Fontinalis antipyretica (Cd in environmentally-relevant concentrations). Whereas phytochelatins were found in the Cd-treated sprouts, no phytochelatins were detected in Fontinalis anitipyretica. PMID:11225681

  19. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  20. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

  1. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  2. Determination of chlorophylls in Taraxacum formosanum by high-performance liquid chromatography-diode array detection-mass spectrometry and preparation by column chromatography.

    PubMed

    Loh, Chin Hoe; Inbaraj, Baskaran Stephen; Liu, Man Hai; Chen, Bing Huei

    2012-06-20

    Taraxacum formosanum, a well-known Chinese herb shown to be protective against hepatic cancer as well as liver and lung damage, may be attributed to the presence of abundant carotenoids and chlorophylls. However, the variety and content of chlorophylls remain uncertain. The objectives of this study were to develop an high-performance liquid chromatography-diode array detection-mass spectrometry method for determination of chlorophylls in T. formosanum and preparation by column chromatography. An HyPURITY C18 column and a gradient mobile phase of water (A), methanol (B), acetonitrile (C), and acetone (D) could resolve 10 chlorophylls and an internal standard Fast Green FCF within 30 min with a flow rate at 1 mL/min and detection at 660 nm. Both chlorophylls a and a' were present in the largest amount (1389.6 μg/g), followed by chlorophylls b and b' (561.2 μg/g), pheophytins a and a' (31.7 μg/g), hydroxychlorophyll b (26.5 μg/g), hydroxychlorophylls a and a' (9.8 μg/g), and chlorophyllides a and a' (0.35 μg/g). A glass column containing 52 g of magnesium oxide-diatomaceous earth (1:3, w/w) could elute chlorophylls with 800 mL of acetone containing 50% ethanol at a flow rate of 10 mL/min. Some new chlorophyll derivatives including chlorophyllide b, pyropheophorbide b, hydroxypheophytin a, and hydroxypheophytin a' were generated during column chromatography but accompanied by a 63% loss in total chlorophylls. Thus, the possibility of chlorophyll fraction prepared from T. formosanum as a raw material for future production of functional food needs further investigation. PMID:22656126

  3. Quantitative liquid chromatography/tandem mass spectrometry determination of chloramphenicol residues in food using sub-2 microm particulate high-performance liquid chromatography columns for sensitivity and speed.

    PubMed

    Kaufmann, Anton; Butcher, Patrick

    2005-01-01

    The use of chloramphenicol (CAP)--a highly effective broad-spectrum antibiotic used in animal husbandry--is banned in many countries. Therefore, a very low minimum required performance limit (MRPL) of 0.3 microg/kg CAP in meat for human consumption has been defined. Analytical methods capable of quantifying and confirming such low residue levels require sophisticated instrumentation. Preferably sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) or gas chromatography/mass spectrometry (GC/MS) methods have been used. This paper suggests the use of sub-2 microm particulate high-performance liquid chromatography (HPLC) columns to gain additional sensitivity and improve resolution as well as speed. Depending on the operating conditions, higher chromatographic resolution and speed can be obtained at the price of a significantly increased operating pressure, requiring dedicated LC equipment. A 3-4-fold overall improvement of the signal-to-noise ratio for CAP was obtained compared to more classical 5 microm particulate HPLC columns. The proposed analytical methodology includes an enzymatic digestion, which liberates glucuronide-bound CAP from kidney tissue. The extracts obtained after an Extrelut clean-up are sufficiently pure to permit routine injection of biological samples into the sub-2 microm particulate HPLC column, without observing rapid deterioration of peak shape or column clogging problems. The time for one chromatographic run was 4.2 min. The described method was validated for two particularly difficult matrices (kidney and honey). Decision limits (CC alpha) were 0.007 microg/kg (honey) und 0.011 microg/kg (kidney), which are significantly below the current MRPL. PMID:16299695

  4. Determination of chlorophylls in Taraxacum formosanum by high-performance liquid chromatography-diode array detection-mass spectrometry and preparation by column chromatography.

    PubMed

    Loh, Chin Hoe; Inbaraj, Baskaran Stephen; Liu, Man Hai; Chen, Bing Huei

    2012-06-20

    Taraxacum formosanum, a well-known Chinese herb shown to be protective against hepatic cancer as well as liver and lung damage, may be attributed to the presence of abundant carotenoids and chlorophylls. However, the variety and content of chlorophylls remain uncertain. The objectives of this study were to develop an high-performance liquid chromatography-diode array detection-mass spectrometry method for determination of chlorophylls in T. formosanum and preparation by column chromatography. An HyPURITY C18 column and a gradient mobile phase of water (A), methanol (B), acetonitrile (C), and acetone (D) could resolve 10 chlorophylls and an internal standard Fast Green FCF within 30 min with a flow rate at 1 mL/min and detection at 660 nm. Both chlorophylls a and a' were present in the largest amount (1389.6 μg/g), followed by chlorophylls b and b' (561.2 μg/g), pheophytins a and a' (31.7 μg/g), hydroxychlorophyll b (26.5 μg/g), hydroxychlorophylls a and a' (9.8 μg/g), and chlorophyllides a and a' (0.35 μg/g). A glass column containing 52 g of magnesium oxide-diatomaceous earth (1:3, w/w) could elute chlorophylls with 800 mL of acetone containing 50% ethanol at a flow rate of 10 mL/min. Some new chlorophyll derivatives including chlorophyllide b, pyropheophorbide b, hydroxypheophytin a, and hydroxypheophytin a' were generated during column chromatography but accompanied by a 63% loss in total chlorophylls. Thus, the possibility of chlorophyll fraction prepared from T. formosanum as a raw material for future production of functional food needs further investigation.

  5. Research on the separation properties of empty-column gas chromatography (EC-GC) and conditions for simulated distillation (SIMDIS).

    PubMed

    Boczkaj, Grzegorz; Kamiński, Marian

    2013-10-01

    Previous studies have revealed it is possible to separate a high-boiling mixture by gas chromatography in empty fused-silica capillary tubing rather than in columns coated with stationary phase. Chromatographic separation occurs solely on the basis of the different boiling points of the substances separated. The high similarity of such separations to those in classic distillation seems advantageous when gas chromatography is used for simulated distillation. This paper presents results from further research on the separation properties of empty fused silica tubing. The efficiency of this chromatographic system has been examined. The usefulness of such conditions has been studied for simulated distillation, i.e. to determine the boiling-point distribution of complex mixtures, mainly petroleum fractions and products, on the basis of their retention relative to reference substances. The results obtained by use of empty-column gas chromatography (EC-GC) and by use of classical simulated distillation columns have been compared for solutes of different polarity. Studies revealed boiling points determined by EC-GC were more accurate than those obtained by the standard method of simulated distillation.

  6. Research on the separation properties of empty-column gas chromatography (EC-GC) and conditions for simulated distillation (SIMDIS).

    PubMed

    Boczkaj, Grzegorz; Kamiński, Marian

    2013-10-01

    Previous studies have revealed it is possible to separate a high-boiling mixture by gas chromatography in empty fused-silica capillary tubing rather than in columns coated with stationary phase. Chromatographic separation occurs solely on the basis of the different boiling points of the substances separated. The high similarity of such separations to those in classic distillation seems advantageous when gas chromatography is used for simulated distillation. This paper presents results from further research on the separation properties of empty fused silica tubing. The efficiency of this chromatographic system has been examined. The usefulness of such conditions has been studied for simulated distillation, i.e. to determine the boiling-point distribution of complex mixtures, mainly petroleum fractions and products, on the basis of their retention relative to reference substances. The results obtained by use of empty-column gas chromatography (EC-GC) and by use of classical simulated distillation columns have been compared for solutes of different polarity. Studies revealed boiling points determined by EC-GC were more accurate than those obtained by the standard method of simulated distillation. PMID:23925798

  7. Analysis of a mixture of polychlorinated biphenyls and chlorinated pesticides in human serum by column fractionation and dual-column capillary gas chromatography with electron capture detection.

    PubMed

    Najam, A R; Korver, M P; Williams, C C; Burse, V W; Needham, L L

    1999-01-01

    An analytical method is presented for precise identification and quantitation of 29 specific polychlorinated biphenyl (PCB) congeners and 15 chlorinated pesticides in human serum. Analyte surrogates PCB 30, PCB 204, 2,2',4,4',5,5'-hexabromo-biphenyl, perthane, alpha-hexachlorocyclohexane, and dichlorobenzophenone were added to each sample. The serum was extracted with an organic solvent and separated by adsorption chromatography into 3 elution fractions for high-resolution gas chromatographic analysis. Each fraction was analyzed by dual-column capillary chromatography followed by electron capture detection. Two capillary columns, DB-5 and DB-1701, with different polarities were used to increase selectivity for each analyte. Quantitation was performed by selecting 2 sets of calibration standard mixtures and 1,2-dichloronaphthalene as an internal standard. Mean recoveries ranged from 39 to 126% for selected analytes and from 31 to 88% for surrogates. Detection limits for specific congeners and pesticides are reported. Typical chromatographic profiles of calibration standard mixtures, as well as a human sample, are illustrated. Verification of each analyte is assessed, and results of analyses of selected human samples and quality control criteria used to ensure data validity also are presented.

  8. Analysis of parabens in cosmetics by low pressure liquid chromatography with monolithic column and chemiluminescent detection.

    PubMed

    Ballesta Claver, J; Valencia, M C; Capitán-Vallvey, L F

    2009-07-15

    This paper presents an application of chromatographic separation based on an ultra-short monolithic column and chemiluminescent detection in an FIA type instrument manifold for the determination of four paraben mixtures: methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP). The separation is achieved in 150 s using two consecutive carriers: first 12% ACN:water that changes 75 s after injection to 27% ACN:water. The detection is based on the oxidation of the hydrolysis product of parabens, p-hydroxybenzoic acid, with Ce(IV) in the presence of Rhodamine 6G which evokes chemiluminescence of sufficient intensity to enable a sensitive determination of these species. After optimization of the variables involved, the analytical method is characterized, displaying the following values for concentration ranges, detection limits and precision, as relative standard deviation at low concentration (0.15 mg l(-1))-MP: from 9.9x10(-7) to 3.3x10(-4)M; 1.9x10(-8); 5.6%; EP: from 9.0x10(-7) to 3.3x10(-4)M; 2.8x10(-8); 3.5%; PP: from 8.3x10(-7) to 9.9x10(-5)M; 2.3x10(-8); 4.2%; and BP: from 7.7x10(-7) to 9.9x10(-5)M; 4.2x10(-8)M; 6.2%. The method was applied and validated satisfactorily for the determination of these parabens in cosmetic samples, comparing the results against a liquid chromatography reference method.

  9. Ion-Exclusion High-Performance Liquid Chromatography of Aliphatic Organic Acids Using a Surfactant-Modified C18 Column.

    PubMed

    Fasciano, Jennifer M; Mansour, Fotouh R; Danielson, Neil D

    2016-07-01

    Ion exclusion chromatography (IELC) of short chain aliphatic carboxylic acids is normally done using a cation exchange column under standard HPLC conditions but not in the ultra-HPLC (UHPLC) mode. A novel IELC method for the separation of this class of carboxylic acids by either HPLC or UHPLC utilizing a C18 column dynamically modified with sodium dodecyl sulfate has been developed. The sample capacity is estimated to be near 10 mM for a 20 µL injection or 0.2 µmol using a 150 × 4.6 mm column. The optimum mobile phase determined for three standard mixtures of organic acids is 1.84 mM sulfuric acid at pH 2.43 and a flow rate of 0.6 mL/min. Under optimized conditions, a HPLC separation of four aliphatic carboxylic acids such as tartaric, malonic, lactic and acetic can be achieved in under 4 min and in <2 min in the UHPLC mode at 2.1 mL/min. A variety of fruit juice and soft drink samples are analyzed. Stability of the column as measured by the retention order of maleic and fumaric acid is estimated to be ∼4,000 column volumes using HPLC and 600 by UHPLC. Reproducible chromatograms are achieved over at least a 2-month period. This study shows that the utility of a C18 column can be easily extended when needed to IELC under either standard or UHPLC conditions.

  10. Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography

    SciTech Connect

    Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.

    2008-10-01

    Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.

  11. Radial heterogeneity of some analytical columns used in high-performance liquid chromatography

    SciTech Connect

    Mriziq, Khaled S; Guiochon, Georges A

    2009-01-01

    An on-column electrochemical microdetector was used to determine accurately the radial distribution of the mobile phase velocity and of the column efficiency at the exit of three common analytical columns, namely a 100 mm x 4.6 mm C18 bonded silica-based monolithic column, a 150 mm x 4.6 mm column packed with 2.7 {micro}m porous shell particles of C18 bonded silica (HALO), and a 150 mm x 4.6 mm column packed with 3 {micro}m fully porous C18 bonded silica particles (LUNA). The results obtained demonstrate that all three columns are not radially homogeneous. In all three cases, the efficiency was found to be lower in the wall region of the column than in its core region (the central core with a radius of 1/3 the column inner radius). The decrease in local efficiency from the core to the wall regions was lower in the case of the monolith (ca. 25%) than in that of the two particle-packed columns (ca. 35-50%). The mobile phase velocity was found to be ca. 1.5% higher in the wall than in the core region of the monolithic column while, in contrast, it was ca. 2.5-4.0% lower in the wall region for the two particle-packed columns.

  12. High performance liquid chromatography column packings with deliberately broadened particle size distribution: relation between column performance and packing structure.

    PubMed

    Liekens, Anuschka; Billen, Jeroen; Sherant, Ron; Ritchie, Harald; Denayer, Joeri; Desmet, Gert

    2011-09-23

    The effect of the addition of 25%, 50% and 75% (weight percent, wt%) of larger particles (resp. 3 and 5 μm) to a commercial batch of 1.9 μm particles has been investigated as an academic exercise to study the effects of particle size distribution on the kinetic performance of packed bed columns in a magnified way. Comparing the performance of the different mixtures in a kinetic plot, it could be irrefutably shown that the addition of larger particles to a commercial batch of small particles cannot be expected to lead to an improved kinetic performance. Whereas the addition of 25 wt% of larger particles still only has a minor negative effect, a significantly deteriorated performance is obtained when 50 or 75 wt% of larger particles are added. In this case, separation impedance number increases up to 200% were observed. Studying the packing structure through computational packing simulations, together with the experimental determination of the external porosity, helped in understanding the obtained results. This showed that small particles tend to settle in the flow-through pores surrounding the larger particles, leading to very high packing densities (external porosities as low as 32% were observed) and also negatively influencing the column permeability as well as the band broadening (because of the broadened flow-through pore size range).

  13. Purification of C70 using charcoal as a stationary phase in a flash chromatography column. Technical report

    SciTech Connect

    Scrivens, W.A.; Cassell, A.M.; Kinsey, K.E.; Tour, J.M.

    1995-06-07

    Described is a method for the purification of C60 and C70 using a flash chromatography column that contains charcoal as the stationary phase. A number of functionalized aromatic solvents are studied and their efficacy for extraction, NMR spectral acquisition, and chromatographic purification of fullerenes is discussed. Ortho-dichlorobenzene was chosen as the best solvent for these applications and examples of its use in the extraction of higher fullerenes (>C84) and in the rapid acquisition of (13)C NMR spectra are given. Finally, single column purification of both C60 and C70 is discussed. Starting with a typical arc-derived mixture of soluble fullerenes, 5.97 g of C60 at >99.9% purity and 1.58 g of C70 at >97% purity were produced in a single column pass.

  14. Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

    PubMed

    Hellstern, Simon; Mutzel, Rupert

    2016-06-01

    We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established. PMID:26892535

  15. Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.

    PubMed

    Barbato, O; Sousa, N M; Klisch, K; Clerget, E; Debenedetti, A; Barile, V L; Malfatti, A; Beckers, J F

    2008-12-01

    The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

  16. Novel strategy using an adsorbent-column chromatography for effective ethanol production from sugarcane or sugar beet molasses.

    PubMed

    Hatano, Ken-ichi; Kikuchi, Satoshi; Nakamura, Yohei; Sakamoto, Hironobu; Takigami, Machiko; Kojima, Yasuyoshi

    2009-10-01

    Molasses-based distilleries generate large volumes of a highly polluted and dark brown-colored wastewater. The present work describes the way in which an adsorbent-column chromatography can effectively remove the colorant and produce biomass ethanol from sugarcane or sugar beet molasses. It was found that the color and chemical oxygen demand of the resulting wastewater was respectively reduced by approximately 87% and 28% as compared with conventional molasses fermentation. Gas chromatography showed that the decolorized molasses maintained good ethanol productivity almost equal to that of the original molasses. Furthermore, it was revealed that the colorant concentrations of about 5 mg ml(-1) in the medium were the most favorable for ethanolic fermentation. In summary, we have concluded that this method is the most effective when the adsorbent chromatography is performed just before molasses fermentation and that the decolorized molasses is an ideal substrate for fuel ethanol production.

  17. Fingerprinting of traditional Chinese medicines on the C18-Diol mixed-mode column in online or offline two-dimensional liquid chromatography on the single column modes.

    PubMed

    Wang, Qing; Tong, Ling; Yao, Lin; Zhang, Peng; Xu, Li

    2016-06-01

    In the present study, a mixed-mode stationary phase, C18-Diol, was applied for fingerprint analysis of traditional Chinese medicines. Hydrophobic, hydrogen bonding and electrostatic interactions were demonstrated to contribute the retention separately or jointly, which endowed the C18-Diol stationary phase with distinct selectivity compared to the bare C18 one. The separation of total alkaloids extracted from Fritillaria hupehensis was compared on the C18-Diol and conventional C18 column with the greater resolving power and better symmetry responses on the former one. Besides, a novel two-dimensional liquid chromatography on the single column (2D-LC-1C) was realized on C18-Diol with the offline mode for the alcohol extract of Fritillaria hupehensis and online mode for Ligusticum chuanxiong Hort. The early co-eluted extracted components with great polarity on the first dimension were reinjected on the same column and well separated on the second dimension. The results exhibited that the two complementary RPLC and HILIC modes on C18-Diol stationary phase enhanced the separation capacity and revealed more abundant chemical information of the sample, which was a powerful tool in analyzing complex herbal medicines. PMID:27031576

  18. Fingerprinting of traditional Chinese medicines on the C18-Diol mixed-mode column in online or offline two-dimensional liquid chromatography on the single column modes.

    PubMed

    Wang, Qing; Tong, Ling; Yao, Lin; Zhang, Peng; Xu, Li

    2016-06-01

    In the present study, a mixed-mode stationary phase, C18-Diol, was applied for fingerprint analysis of traditional Chinese medicines. Hydrophobic, hydrogen bonding and electrostatic interactions were demonstrated to contribute the retention separately or jointly, which endowed the C18-Diol stationary phase with distinct selectivity compared to the bare C18 one. The separation of total alkaloids extracted from Fritillaria hupehensis was compared on the C18-Diol and conventional C18 column with the greater resolving power and better symmetry responses on the former one. Besides, a novel two-dimensional liquid chromatography on the single column (2D-LC-1C) was realized on C18-Diol with the offline mode for the alcohol extract of Fritillaria hupehensis and online mode for Ligusticum chuanxiong Hort. The early co-eluted extracted components with great polarity on the first dimension were reinjected on the same column and well separated on the second dimension. The results exhibited that the two complementary RPLC and HILIC modes on C18-Diol stationary phase enhanced the separation capacity and revealed more abundant chemical information of the sample, which was a powerful tool in analyzing complex herbal medicines.

  19. One-step purification of lactoperoxidase from bovine milk by affinity chromatography.

    PubMed

    Atasever, Ali; Ozdemir, Hasan; Gulcin, Ilhami; Irfan Kufrevioglu, O

    2013-01-15

    Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC(50) of 0.848.10(-5)M. The K(i) for sulphanilamide was determined to be 3.57.10(-5)M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R(z) (A(412)/A(280)) value. The R(z) value for the purified LPO was found to be 0.7. Maximum binding was achieved and K(m) and V(max) values were determined.

  20. Exogenous factors contributing to column bed heterogeneity: Part 1: Consequences of 'air' injections in liquid chromatography.

    PubMed

    Samuelsson, Jörgen; Fornstedt, Torgny; Shalliker, Andrew

    2015-08-01

    It has been shown that not only the packing homogeneity, but also factors external to the column bed, such as, frits and distributors can have important effects on the column performance. This current communication is the first in a series focusing on the impact of exogenous factors on the column bed heterogeneity. This study is based on several observations by us and others that chromatographic runs often, for technical reasons, include more or less portions of air in the injections. It is therefore extremely important to find out the impact of air on the column performance, the reliability of the results derived from analyses where air was injected, and the effect on the column homogeneity. We used a photographic approach for visualising the air transport phenomena, and found that the air transport through the column is comprised of many different types of transport phenomena, such as laminal flow, viscous fingering like flows, channels and bulbs, and pulsations. More particularly, the air clouds within the column definitely interact in the adsorption, i.e. mobile phase adsorbed to the column surface is displaced. In addition, irrespective of the type of air transport phenomena, the air does not penetrate the column homogeneously. This process is strongly flow dependent. In this work we study air transport both in an analytical scale and a semi-prep column.

  1. Identification of IgG alloantibodies in patients with high-titer IgM cold agglutinins by serum/plasma affinity chromatography.

    PubMed

    Stahl, D; Kreft, H; Hack, H; Schraven, B; Roelcke, D

    1997-01-01

    The detection of IgG alloantibodies in the presence of high-titer cold autoagglutinins (CAs) can be extremely difficult, especially under pressure of time when transfusion of red blood cells is urgently needed. Here we demonstrate that IgG alloantibodies in the presence of high-titer IgM CAs can be easily detected by quantitative IgG purification from serum or plasma by affinity chromatography. In comparison with the routinely used methods for IgG alloantibody identification, affinity chromatography shows better or identical results and is the method leading to results most rapidly.

  2. Simple column-switching ion chromatography method for determining eight monosaccharides and oligosaccharides in honeydew and nectar.

    PubMed

    Ni, Chengzhu; Zhu, Binhe; Wang, Nani; Wang, Muhua; Chen, Suqing; Zhang, Jiajie; Zhu, Yan

    2016-03-01

    Honeydew is excreted by aphids as a sweet waste and nectar is floral honey. Honeydew and nectar are complicated samples which consist of various sugars and amino acids. In this work, a simple ion chromatography with column-switching method was developed for the simultaneous analysis of 8 monosaccharides and oligosaccharides in honeydew and nectar. A reversed-phase column was used as a pretreatment column to eliminate organics on-line and sugars were eluted from a collection loop to analytical column by using column-switching technique. This method showed good linearity (r⩾0.9994) and afforded low limits of detection ranging from 1.55 to 10.17μgL(-1) for all the analytes. Recoveries ranged from 95% to 105% and repeatability results were acceptable with relative standard deviation of less than 3.21% (n=6). This method was successfully applied to quantification of these sugars in honeydew and nectar. These results showed honeydew had much more oligosaccharides than nectar.

  3. Simultaneous determination of peroxydisulfate and conventional inorganic anions by ion chromatography with the column-switching technique.

    PubMed

    Huang, Zhongping; Ni, Chengzhu; Wang, Fengli; Zhu, Zuoyi; Subhani, Qamar; Wang, Muhua; Zhu, Yan

    2014-02-01

    The application of ion chromatography with the column-switching technique for the simultaneous analysis of peroxydisulfate and conventional inorganic anions in a single run is described. With this method, conventional inorganic anions were separated by consecutive elution through both the guard column and separation column, but peroxydisulfate that only passed through the guard column had a good peak shape and short retention time. A series of standard solutions consisting of target anions of various concentrations from 0.01 to 75 mg/L were analyzed, with a correlation coefficient (r) ≥ 0.9990. The limits of detection were in the range of 0.49-9.84 μg/L based on the S/N of 3 and a 25 μL injection volume. RSDs for retention time, peak area, and peak height were all <1.77%. A spiking study was performed with satisfactory recoveries between 97.6 and 103.4% for all anions. The quantitative determination of peroxydisulfate and conventional inorganic anions in surface waters was accomplished within 18 min by this column-switching technique.

  4. 3D printed titanium micro-bore columns containing polymer monoliths for reversed-phase liquid chromatography.

    PubMed

    Gupta, Vipul; Talebi, Mohammad; Deverell, Jeremy; Sandron, Sara; Nesterenko, Pavel N; Heery, Brendan; Thompson, Fletcher; Beirne, Stephen; Wallace, Gordon G; Paull, Brett

    2016-03-01

    The potential of 3D selective laser melting (SLM) technology to produce compact, temperature and pressure stable titanium alloy chromatographic columns is explored. A micro bore channel (0.9 mm I.D. × 600 mm long) was produced within a 5 × 30 × 30 mm titanium alloy (Ti-6Al-4V) cuboid, in form of a double handed spiral. A poly(butyl methacrylate-co-ethyleneglycoldimethacrylate) (BuMA-co-EDMA) monolithic stationary phase was thermally polymerised within the channel for application in reversed-phase high-performance liquid chromatography. The prepared monolithic column was applied to the liquid chromatographic separation of intact proteins and peptides. Peak capacities of 69-76 (for 6-8 proteins respectively) were observed during isothermal separation of proteins at 44 °C which were further increased to 73-77 using a thermal step gradient with programmed temperature from 60 °C to 35 °C using an in-house built direct-contact heater/cooler platform based upon matching sized Peltier thermoelectric modules. Rapid temperature gradients were possible due to direct-contact between the planar metal column and the Peltier module, and the high thermal conductivity of the titanium column as compared to a similar stainless steel printed column. The separation of peptides released from a digestion of E.coli was also achieved in less than 35 min with ca. 40 distinguishable peaks at 210 nm. PMID:26873472

  5. 3D printed titanium micro-bore columns containing polymer monoliths for reversed-phase liquid chromatography.

    PubMed

    Gupta, Vipul; Talebi, Mohammad; Deverell, Jeremy; Sandron, Sara; Nesterenko, Pavel N; Heery, Brendan; Thompson, Fletcher; Beirne, Stephen; Wallace, Gordon G; Paull, Brett

    2016-03-01

    The potential of 3D selective laser melting (SLM) technology to produce compact, temperature and pressure stable titanium alloy chromatographic columns is explored. A micro bore channel (0.9 mm I.D. × 600 mm long) was produced within a 5 × 30 × 30 mm titanium alloy (Ti-6Al-4V) cuboid, in form of a double handed spiral. A poly(butyl methacrylate-co-ethyleneglycoldimethacrylate) (BuMA-co-EDMA) monolithic stationary phase was thermally polymerised within the channel for application in reversed-phase high-performance liquid chromatography. The prepared monolithic column was applied to the liquid chromatographic separation of intact proteins and peptides. Peak capacities of 69-76 (for 6-8 proteins respectively) were observed during isothermal separation of proteins at 44 °C which were further increased to 73-77 using a thermal step gradient with programmed temperature from 60 °C to 35 °C using an in-house built direct-contact heater/cooler platform based upon matching sized Peltier thermoelectric modules. Rapid temperature gradients were possible due to direct-contact between the planar metal column and the Peltier module, and the high thermal conductivity of the titanium column as compared to a similar stainless steel printed column. The separation of peptides released from a digestion of E.coli was also achieved in less than 35 min with ca. 40 distinguishable peaks at 210 nm.

  6. Computational fluid dynamics simulations yielding guidelines for the ideal internal structure of monolithic liquid chromatography columns.

    PubMed

    Gzil, P; Baron, G V; Desmet, G

    2003-04-01

    A theoretical calculation of the separation performance of a (hypothetical) micro-structured monolithic LC column is presented, confirming that the polydispersity effect in parallel bundle columns can theoretically be eliminated to a very large extent by radially redistributing the mobile phase fluid at regular intervals. It is demonstrated that the flow can be redistributed in such a way that the advantage coming from the suppression of the polydispersity effect largely exceeds the losses caused by the additional pressure-drop and band broadening. The presently considered micro-structured column would allow to perform N > 100,000 plate separations in a few hundred of seconds, i.e., about an order of magnitude faster than the best possible packed bed and monolithic HPLC columns, while offering the same mass loadability. This clearly demonstrates that the currently available LC columns are still far away from the absolute resolution limit of the ideal, fully optimised LC column.

  7. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    PubMed Central

    Lu, Lina; Qi, Zhijun; Zhang, Jiwen; Wu, Wenjun

    2015-01-01

    Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed. PMID:25996604

  8. Purification of Hemoglobin from Red Blood Cells using Tangential Flow Filtration and Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Elmer, Jacob; Harris, David; Palmer, Andre F.

    2011-01-01

    Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are examined and compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter. PMID:21195679

  9. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    PubMed

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. PMID:26657801

  10. Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography.

    PubMed

    Bartnicki, Filip; Kowalska, Ewa; Pels, Katarzyna; Strzalka, Wojciech

    2015-10-30

    Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin. PMID:26427325

  11. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography.

    PubMed

    Lu, Lina; Qi, Zhijun; Zhang, Jiwen; Wu, Wenjun

    2015-05-01

    Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed. PMID:25996604

  12. Multi-Parameter Cell Affinity Chromatography: Separation and Analysis in a Single Microfluidic Channel

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2012-01-01

    The ability to sort and capture more than one cell type from a complex sample will enable a wide variety of studies of cell proliferation, death, and the analysis of disease states. In this work, we integrated a pneumatic actuated control layer to an affinity separation layer to create different antibody coating regions on the same fluidic channel. The comparison of different antibody capture capabilities to the same cell line was demonstrated by flowing Ramos cells through anti-CD19 and anti-CD71 coated regions in the same channel, respectively. It was determined that cell capture density on anti-CD19 region was 2.44±0.13 times higher than on anti-CD71 coated region. This approach can be used to test different affinity molecules for selectivity and capture efficiency using a single cell line in one separation. Selective capture of Ramos and HuT 78 cells from a mixture was also demonstrated using two antibody regions in the same channel. Greater than 90% purity was obtained on both capture areas in both continuous flow and stop flow separation modes. A four-region antibody coated device was then fabricated to study the simultaneous, serial capture of three different cell lines. In this case the device showed effective capture of cells in a single separation channel, opening up the possibility of multiple cell sorting. Multi-parameter sequential blood sample analysis was also demonstrated with high capture specificity (>97% for both CD19+ and CD4+ leukocytes). The chip can also be used to selectively treat cells after affinity separation. PMID:22958145

  13. Optimized use of a 50mum ID secondary column in comprehensive two-dimensional gas chromatography-mass spectrometry.

    PubMed

    Tranchida, Peter Quinto; Purcaro, Giorgia; Fanali, Chiara; Dugo, Paola; Dugo, Giovanni; Mondello, Luigi

    2010-06-18

    The objective of the present research is directed towards the optimized use of a 50microm ID secondary column, in a comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry (GCxGC-qMS) system. The analytical aim was achieved by exploiting a split-flow GCxGC approach, and a rapid-scanning qMS instrument. The stationary phase combination consisted of an apolar (silphenylene polymer) 30mx0.25mm ID column, linked by means of a Y-union, to an MS-connected 1mx0.05mm ID polar one [poly(ethyleneglycol)], and to a 0.20mx0.05mm ID uncoated capillary segment; the latter was connected to a manually operated split-valve. It will be herein demonstrated that the split-flow GCxGC approach, successfully employed in previous H(2)-based, flame ionization detection experiments, provides equally satisfactory results using mass spectrometric detection and helium as carrier gas. An optimized split-flow GCxGC-qMS method was developed and exploited for the analysis of a perfume sample. The results attained were compared with those observed using the same analytical column combination, but with no flow-splitting. It was found that it is not convenient to employ a 50microm ID secondary column in a conventional GCxGC-MS instrument. On the contrary, the use a 50microm ID secondary column, in a split-flow, twin-oven system, provided a good performance. A recently developed comprehensive chromatography software was used for data processing.

  14. Effects of temperature and mobile-phase composition on retentions in column liquid chromatography. [Hydroxyl-terminated polybutadiene

    SciTech Connect

    Hsu, A.

    1982-08-01

    Binary-additive mobile phase systems were investigated for a more exact control over chromatography. If the mobile phase was nonpolar binary solvents (e.g., n-hexane and dichloromethane), trace amounts of THF and/or ACN could be added to control solute retentions as a function of solvent composition. Temperature also plays an important role in the coverage of modifier molecules on the surface of the adsorbent. Higher efficiencies were obtained at lower temperatures. For chromatographic systems having large extra-column volumes, higher column temperature will also reduce column efficiency. Solvophobic effects and polar-group selectivity control the chromatographic behavior of phenols in reversed-phase liquid chromatography (RPLC). The former effect predominates with the THF system, and the latter with the MeOH system. Substituent groups decrease the hydrophobic effect of phenols in aqueous media. However, owing to steric effects, ortho-substituted phenols usually eluted later. Separation of the phenols was also found with 35% MeOH/H/sub 2/O eluent. Application of window diagrams led to baseline separation with 22% THF/H/sub 2/O. Optimum column temperature was 42/sup 0/C with a Zorbax C8 microbore column. The enthalpy-entropy compensation principle was applied to the retention data observed with reversed-phase systems. Relative retention data were found to provide a more consistent compensation temperature. RPLC was shown to be useful in the separation of high molecular-weight compounds such as hydroxyl-terminated polybutadiene (HTPB). HMDS-treated HTPB eluted without difficulty from silical gel. Sample overload can provide unique separations in GPC. (DLC)

  15. Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel.

    PubMed

    Beyaztaş, Serap; Arslan, Oktay

    2015-06-01

    A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150 kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60 °C and incubated for 60 min. These results indicated that the enzyme was heat stable. PMID:25089709

  16. Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors

    PubMed Central

    Kuester, Miriam; Becker, Gero L.; Hardes, Kornelia; Lindberg, Iris; Steinmetzer, Torsten; Than, Manuel E.

    2013-01-01

    In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied – studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)2-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members. PMID:21875402

  17. Isolation and purification of diastereoisomeric flavonolignans from silymarin by binary-column recycling preparative high-performance liquid chromatography.

    PubMed

    Zhao, Weiquan; Yang, Guang; Zhong, Fanyi; Yang, Nan; Zhao, Xin; Qi, Yunpeng; Fan, Guorong

    2014-09-01

    Silymarin extracted from Silybum marianum (L.) Gaertn consists of a large number of flavonolignans, of which diastereoisomeric flavonolignans including silybin A and silybin B, and isosilybin A and isosilybin B are the main bioactive components, whose preparation from the crude extracts is still a difficult task. In this work, binary-column recycling preparative high-performance liquid chromatography systems without sample loop trapping, where two columns were switched alternately via one or two six-port switching valves, were established and successfully applied to the isolation and purification of the four diastereoisomeric flavonolignans from silymarin. The proposed system showed significant advantages over conventional preparative high-performance liquid chromatography with a single column in increasing efficiency and reducing the cost. To obtain the same amounts of products, the proposed system spends only one tenth of the time that the conventional system spends, and needs only one eleventh of the solvent that the conventional system consumes. Using the proposed system, the four diastereoisomers were successfully isolated from silymarin with purities over 98%.

  18. Robust design of adenovirus purification by two-column, simulated moving-bed, size-exclusion chromatography.

    PubMed

    Nestola, Piergiuseppe; Silva, Ricardo J S; Peixoto, Cristina; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

    2015-11-10

    A simple, yet efficient, two-column simulated moving-bed (2CSMB) process for purifying adenovirus serotype 5 (Ad5) by size-exclusion chromatography (SEC) is presented and validated experimentally, and a general procedure for its robust design under parameter uncertainty is described. The pilot-scale run yielded a virus recovery of 86 percent and DNA and HCP clearances of 90 and 89 percent, respectively, without any fine tuning of the operating parameters. This performance compares very favorably against that of single-column batch chromatography for the same volume of size-exclusion resin. To improve the robustness of the 2CSMB-SEC process the best set of operating parameters is selected only among candidate solutions that are robust feasible, that is, remain feasible for all parameter perturbations within their uncertainty intervals. This robust approach to optimal design replaces the nominal problem by a worst case problem. Computational tractability is ensured by formulating the robust design problem with only the vertices of the uncertainty region that have the worst effect on the product purity and recovery. The robust design is exemplified on the case where the column volume and interparticle porosity are subject to uncertainty. As expected, to increase the robustness of the 2CSMB-SEC process it is necessary to reduce its productivity and increase its solvent consumption. Nevertheless, the design solution given by our robust approach is the least detrimental of all feasible operating conditions for the 2CSMB-SEC process.

  19. The Plasma Membrane Ca(2+) ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.

    PubMed

    Niggli, Verena; Carafoli, Ernesto

    2016-01-01

    Plasma membrane Ca(2+) ATPases (PMCA pumps) are key regulators of cytosolic Ca(2+) in eukaryotes. They extrude Ca(2+) from the cytosol, using the energy of ATP hydrolysis and operate as Ca(2+)-H(+) exchangers. They are activated by the Ca(2+)-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca(2+)-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form. PMID:26695022

  20. Analysis of glipizide binding to normal and glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-07-01

    In diabetes, the elevated levels of glucose in the bloodstream can result in the nonenzymatic glycation of proteins such as human serum albumin (HSA). This type of modification has been shown to affect the interactions of some drugs with HSA, including several sulfonylurea drugs that are used to treat type II diabetes. This study used high-performance affinity chromatography (HPAC) to examine the interactions of glipizide (i.e., a second-generation sulfonylurea drug) with normal HSA or HSA that contained various levels of in vitro glycation. Frontal analysis indicated that glipizide was interacting with both normal and glycated HSA through two general groups of sites: a set of relatively strong interactions and a set of weaker interactions with average association equilibrium constants at pH 7.4 and 37 °C in the range of 2.4-6.0 × 10(5) and 1.7-3.7 × 10(4) M(-1), respectively. Zonal elution competition studies revealed that glipizide was interacting at both Sudlow sites I and II, which were estimated to have affinities of 3.2-3.9 × 10(5) and 1.1-1.4 × 10(4) M(-1). Allosteric effects were also noted to occur for this drug between the tamoxifen site and the binding of R-warfarin at Sudlow site I. Up to an 18% decrease in the affinity for glipizide was observed at Sudlow site I ongoing from normal HSA to glycated HSA, while up to a 27% increase was noted at Sudlow site II. This information should be useful in indicating how HPAC can be used to investigate other drugs that have complex interactions with proteins. These results should also be valuable in providing a better understanding of how glycation may affect drug-protein interactions and the serum transport of drugs such as glipizide during diabetes. PMID:25912461

  1. Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography.

    PubMed

    Tachibana, Kouichi; Nakamura, Sachiko; Wang, Han; Iwasaki, Hiroko; Tachibana, Kahori; Maebara, Kanako; Cheng, Lamei; Hirabayashi, J; Narimatsu, H

    2006-01-01

    Jacalin, a lectin from the jackfruit Artocarpus integrifolia, has been known as a valuable tool for specific capturing of O-glycoproteins such as mucins and IgA1. Though its sugar-binding preference for T/Tn-antigens is well established, its detailed specificity has not been elucidated. In this study, we prepared a series of mucin-type glycopeptides using human glycosyltransferases, that is, ST6GalNAc1, Core1Gal-T1 and -T2, beta3Gn-T6, and Core2GnT1, and investigated their binding to immobilized Jacalin by frontal affinity chromatography (FAC). As a result, consistent with the previous observation, Jacalin showed high affinity for T-antigen (Core1) and Tn-antigen (alpha N-acetylgalactosamine)-attached peptides. Furthermore, we here show as novel findings that (1) Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides. The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides. In conclusion, Jacalin binds to a GalNAcalpha1-peptide, in which C6-OH of alphaGalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAcalpha1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn). These findings provide useful information when applying jacalin for functional analysis of mucin-type glycoproteins and glycopeptides.

  2. Quasi-adiabatic vacuum-based column housing for very high-pressure liquid chromatography.

    PubMed

    Gritti, Fabrice; Gilar, Martin; Jarrell, Joseph A

    2016-07-22

    A prototype vacuum-based (10(-6)Torr) column housing was built to thermally isolate the chromatographic column from the external air environment. The heat transfer mechanism is solely controlled by surface radiation, which was minimized by wrapping the column with low-emissivity aluminum tape. The adiabaticity of the column housing was quantitatively assessed from the measurement of the operational pressure and fluid temperature at the outlet of a 2.1mm×100mm column (sub-2 μm particles). The pressure drop along the column was raised up to 1kbar. The enthalpy balance of the eluent (water, acetonitrile, and one water/acetonitrile mixture, 70/30, v/v) showed that less than 1% of the viscous heat generated by friction of the fluid against the packed bed was lost to the external air environment. Such a vacuum-based column oven minimizes the amplitude of the radial temperature gradients across the column diameter and maximizes its resolving power.

  3. Planar gas chromatography column on glass plate with nanodispersed silica as the stationary phase

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    The paper presents the GC column in the plane of the glass plate with the adsorption layer nanodispersed silica. Created gas chromatographic column allows to separate a mixture of five alkanes from pentane to nonane in isothermal (90 ° C) mode less than one minute.

  4. Effects of a malfunctional column on conventional and FeedCol-simulated moving bed chromatography performance.

    PubMed

    Song, Ji-Yeon; Oh, Donghoon; Lee, Chang-Ha

    2015-07-17

    The effects of a malfunctional column on the performance of a simulated moving bed (SMB) process were studied experimentally and theoretically. The experimental results of conventional four-zone SMB (2-2-2-2 configuration) and FeedCol operation (2-2-2-2 configuration with one feed column) with one malfunctional column were compared with simulation results of the corresponding SMB processes with a normal column configuration. The malfunctional column in SMB processes significantly deteriorated raffinate purity. However, the extract purity was equivalent or slightly improved compared with the corresponding normal SMB operation because the complete separation zone of the malfunctional column moved to a lower flow rate range in zones II and III. With the malfunctional column configuration, FeedCol operation gave better experimental performance (up to 7%) than conventional SMB operation because controlling product purity with FeedCol operation was more flexible through the use of two additional operating variables, injection time and injection length. Thus, compared with conventional SMB separation, extract with equivalent or slightly better purity could be produced from FeedCol operation even with a malfunctional column, while minimizing the decrease in raffinate purity (less than 2%).

  5. Quasi-adiabatic vacuum-based column housing for very high-pressure liquid chromatography.

    PubMed

    Gritti, Fabrice; Gilar, Martin; Jarrell, Joseph A

    2016-07-22

    A prototype vacuum-based (10(-6)Torr) column housing was built to thermally isolate the chromatographic column from the external air environment. The heat transfer mechanism is solely controlled by surface radiation, which was minimized by wrapping the column with low-emissivity aluminum tape. The adiabaticity of the column housing was quantitatively assessed from the measurement of the operational pressure and fluid temperature at the outlet of a 2.1mm×100mm column (sub-2 μm particles). The pressure drop along the column was raised up to 1kbar. The enthalpy balance of the eluent (water, acetonitrile, and one water/acetonitrile mixture, 70/30, v/v) showed that less than 1% of the viscous heat generated by friction of the fluid against the packed bed was lost to the external air environment. Such a vacuum-based column oven minimizes the amplitude of the radial temperature gradients across the column diameter and maximizes its resolving power. PMID:27324623

  6. [Rapid determination of propranolol enantiomers in rat plasma by column-switching-high performance liquid chromatography].

    PubMed

    Wu, Xiaoyu; Wang, Rong; Xie, Hua; Wang, Jianfeng; Jia, Zhengping; Zhang, Qiang; Wang, Xianhua

    2011-12-01

    A high performance liquid chromatographic (HPLC) method with column-switching was developed and validated for rapid determination of two propranolol enantiomers in rat plasma. The column of restricted-access media was used as a pre-treatment column and a Chiralcel OD-RH was used as analytical column. The plasma samples were injected directly into the pretreatment column to remove plasma protein and endogenous constituents as well as to retent the propranolol enantiomers in the column using the mobile phase of borate buffer (pH 8.5)-methanol (95:5, v/v) at the flow rate of 1.0 mL/min. Then the propranolol enantiomers were transferred to the Chiralcel OD-RH column using the mobile phase of isopropanol-ethanol-0.2 mmol/L borate buffer (pH 8.5) (30: 30: 40, v/v/v) at a flow rate of 0.8 mL/min by column-switching technology. The column-switching time was 3.0 min, the used wavelength was 293 nm and the column temperature was set at 25 degrees C. The calibration curve showed excellent linear relationship (r = 0.999 5) in the concentration range from 25 mg/L to 500 mg/L for propranolol enantiomers in plasma. The intra-day and inter-day assay precisions and accuracies were well and the relative standard deviations (RSDs) were less than 5%. The average recoveries (n = 6) of the two enantiomers at 3 spiked levels were from 97.89% to 101.56%. All the values of the method validation were within the generally accepted criteria for biological sample analysis. The results show that the method is convenient, quick, sensitive and accurate. The method was successfully applied in the determination of propranolol enantiomers in rat blood pharmacokinetics study.

  7. Investigation of a new core-shell particle column for ion-pair reversed-phase liquid chromatography analysis of oligonucleotides.

    PubMed

    Biba, Mirlinda; Welch, Christopher J; Foley, Joe P

    2014-08-01

    A new core-shell particle column showed excellent performance and durability for separation of short (∼21-mer) ribonucleic acid (RNA) oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC). Previously investigated core-shell C18 columns showed excellent peak shapes and separations of closely eluting impurities by IP-RPLC. However, these columns showed only modest long-term stability at the neutral pH and elevated column temperatures of ≥60°C, typically used for IP-RPLC analysis of oligonucleotides. The newly introduced SunShell C18 column provided separations comparable to the previously evaluated core-shell columns, but with significantly improved long-term column stability when operated at neutral pH and elevated column temperature.

  8. Ion-Exclusion High-Performance Liquid Chromatography of Aliphatic Organic Acids Using a Surfactant-Modified C18 Column.

    PubMed

    Fasciano, Jennifer M; Mansour, Fotouh R; Danielson, Neil D

    2016-07-01

    Ion exclusion chromatography (IELC) of short chain aliphatic carboxylic acids is normally done using a cation exchange column under standard HPLC conditions but not in the ultra-HPLC (UHPLC) mode. A novel IELC method for the separation of this class of carboxylic acids by either HPLC or UHPLC utilizing a C18 column dynamically modified with sodium dodecyl sulfate has been developed. The sample capacity is estimated to be near 10 mM for a 20 µL injection or 0.2 µmol using a 150 × 4.6 mm column. The optimum mobile phase determined for three standard mixtures of organic acids is 1.84 mM sulfuric acid at pH 2.43 and a flow rate of 0.6 mL/min. Under optimized conditions, a HPLC separation of four aliphatic carboxylic acids such as tartaric, malonic, lactic and acetic can be achieved in under 4 min and in <2 min in the UHPLC mode at 2.1 mL/min. A variety of fruit juice and soft drink samples are analyzed. Stability of the column as measured by the retention order of maleic and fumaric acid is estimated to be ∼4,000 column volumes using HPLC and 600 by UHPLC. Reproducible chromatograms are achieved over at least a 2-month period. This study shows that the utility of a C18 column can be easily extended when needed to IELC under either standard or UHPLC conditions. PMID:27006111

  9. Rapid determination of Papaver somniferum alkaloids in process streams using monolithic column high-performance liquid chromatography with chemiluminescence detection.

    PubMed

    Costin, Jason W; Lewis, Simon W; Purcell, Stuart D; Waddell, Lucy R; Francis, Paul S; Barnett, Neil W

    2007-07-30

    We have combined high-performance liquid chromatography (HPLC) separations using a monolithic column with acidic potassium permanganate and tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection in a rapid and highly sensitive method to monitor the process of extracting opiate alkaloids from Papaver somniferum. Due to the high flow rates allowed with the monolithic column and the inherent selectivity of the chemiluminescence reactions, the four predominant alkaloids--morphine, codeine, oripavine and thebaine--were determined in less than 2 min. The results obtained with numerous process samples compared favourable with those of the standard HPLC methodology. Limits of detection were 1x10(-10) M, 5x10(-10) M, 5x10(-10) M and 1x10(-9) M, for morphine, codeine, oripavine and thebaine, respectively.

  10. Detection of radiation-induced hydrocarbons in Camembert irradiated before and after the maturing process-comparison of florisil column chromatography and on-line coupled liquid chromatography-gas chromatography

    SciTech Connect

    Schulzki, G.; Spiegelberg, A.; Schreiber, G.A.

    1995-02-01

    The influence of the maturing process on the detection of radiation-induced volatile hydrocarbons in the fat of Camembert has been investigated. Two analytical methods for separation of the hydrocarbon fraction from the lipid were applied: Florisil column chromatography with subsequent gas chromatographic-mass spectrometric (GC-MS) determination as well as on-line coupled liquid chromatography-GC-MS. The maturing process had no influence on the detection of radiation-induced volatiles. Comparable results were achieved with both analytical methods. However, preference is given to the more effective on-line coupled LC-GC method. 17 refs., 5 figs., 2 tabs.

  11. Multi-temperature gas chromatography using isothermal columns in series. Technical report

    SciTech Connect

    Spencer, W.A.; Rogers, L.B.

    1980-08-11

    A computer-controlled three-oven gas chromatograph having a multi-position stream-switching valve has been used first to divide a wide-boiling mixture of n-alkanes into three cuts and then to fractionate each cut using a packed column at a different temperature. After initial injection of the sample into the highest temperature column, the low boilers were switched to a low temperature oven. Then, the mid-boilers were switched to a column at an intermediate temperature. For routine repetitive analyses, the multi-oven approach offers an attractive alternative.

  12. Direct enantioseparation of nitrogen-heterocyclic pesticides on cellulose-based chiral column by high-performance liquid chromatography.

    PubMed

    Chai, Tingting; Yang, Wenwen; Qiu, Jing; Hou, Shicong

    2015-01-01

    The enantiomeric separation of eight pesticides including bitertanol (), diclobutrazol (), fenbuconazole (), triticonazole (), imazalil (), triapenthenol (), ancymidol (), and carfentrazone-ethyl () was achieved, using normal-phase high-performance liquid chromatography on two cellulosed-based chiral columns. The effects of isopropanol composition from 2% to 30% in the mobile phase and column temperature from 5 to 40 °C were investigated. Satisfactory resolutions were obtained for bitertanol (), triticonazole (), imazalil () with the (+)-enantiomer eluted first and fenbuconazole () with the (-)-enantiomer eluted first on Lux Cellulose-2 and Lux Cellulose-3. (+)-Enantiomers of diclobutrazol () and triapenthenol () were first eluted on Lux Cellulose-2. (-)-Carfentrazone-ethyl () were eluted first on Lux Cellulose-2 and Lux Cellulose-3 with incomplete separation. Reversed elution orders were obtained for ancymidol (7). (+)-Ancymidol was first eluted on Lux Cellulose-2 while on Lux Cellulose-3 (-)-ancymidol was first eluted. The results of the elution order at different column temperatures suggested that column temperature did not affect the optical signals of the enantiomers. These results will be helpful to prepare and analyze individual enantiomers of chiral pesticides.

  13. High resolution reversed phase analysis of recombinant monoclonal antibodies by ultra-high pressure liquid chromatography column coupling.

    PubMed

    Fekete, Szabolcs; Dong, Michael W; Zhang, Taylor; Guillarme, Davy

    2013-09-01

    Monoclonal antibodies (mAbs) represent one of the fastest growing areas of new drug development. However, their analytical characterization is complex and generally requires an array of orthogonal analytical techniques. Reversed phase liquid chromatography is a valuable strategy due to its high resolving power and straightforward compatibility to mass spectrometry. The present study demonstrates that high peak capacity can be attained with intact mAb of ~150 kDa, reduced mAb fragments of ~25-50 kDa and also digested mAb generating numerous peptides of ~0.5-3 kDa. Several long columns packed with fully porous wide-pore sub-2 μm particles were coupled in series to evaluate the effect of column length on peak capacity. By using three columns of 150 mm length, a mobile phase temperature of 80 °C and a gradient time of around 20 min, peak capacities of 117 and 128 were obtained for a commercial intact mAb and its reduced mAb fragments, respectively. On the other hand, when peptide mapping was performed at 50 °C, with a gradient time of 270 min and a column length of 450 mm, a peak capacity of more than 700 was achieved.

  14. Separation of beta-human chorionic gonadotropin and immunoglobulin G by a miniaturized size exclusion chromatography column

    NASA Astrophysics Data System (ADS)

    Yang, Yongmo; Chae, Junseok

    2009-04-01

    This report describes a miniaturized size exclusion chromatography column that effectively preseparates raw samples for medical point-of-care testing (POCT) devices. The minicolumn is constructed of polydimethylsiloxane fabricated on a glass slide. The minicolumn separates 300 ng/ml of beta-human chorionic gonadotropin (β-hCG) from an immunoglobulin G (IgG)-rich solution (100 μg/ml) in 7.7 min, with 2.23 resolution and 0.018 mm plate height. The complete analyte discrimination shows potential for the sample preparation stage of POCT devices for cancer screening, prognosis, and monitoring.

  15. Direct probing of chromatography columns by laser-induced fluorescence. Technical progress report, September 1, 1989--February 28, 1993

    SciTech Connect

    McGuffin, V.L.

    1992-12-07

    This report summarizes the progress and accomplishments of this research project from September 1, 1989 to February 28, 1993. During this period, we have accomplished all of the primary scientific objectives of the research proposal: (1) constructed and evaluated a laser-induced fluorescence detection system that allows direct examination of the chromatographic column, (2) examined nonequilibrium processes that occur upon solute injection and elution, (3) examined solute retention in liquid chromatography as a function of temperature and pressure, (4) examined solute zone dispersion in liquid chromatography as a function of temperature and pressure, and (5) developed appropriate theoretical models to describe these phenomena. In each of these studies, substantial knowledge has been gained of the fundamental processes that are responsible for chromatographic separations. In addition to these primary research objectives, we have made significant progress in three related areas: (1) examined pyrene as a fluorescent polarity probe insupercritical fluids and liquids as a function of temperature and pressure, (2) developed methods for the class-selective identification of polynuclear aromatic hydrocarbons in coal-derived fluids by microcolumn liquid chromatography with fluorescence quenching detection, and (3) developed methods for the determination of saturated and unsaturated (including omega-3) fatty acids in fish oil extracts by microcolumn liquid chromatography with laser-induced fluorescence detection. In these studies, the advanced separation and detection techniques developed in our laboratory are applied to practical problems of environmental and biomedical significance.

  16. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  17. Determination of carotenoids in Taraxacum formosanum by HPLC-DAD-APCI-MS and preparation by column chromatography.

    PubMed

    Kao, T H; Loh, C H; Inbaraj, B Stephen; Chen, B H

    2012-07-01

    The objectives of this study were to determine the variety and content of carotenoids in Taraxacum formosanum, a traditional Chinese herb possessing vital biological activities, by developing an HPLC-DAD-APCI-MS method and a preparative column chromatographic method for carotenoid isolation. A total of 25 carotenoids were resolved within 66 min by employing a YMC C30 column and a gradient mobile phase of methanol-acetonitrile-water (79:14:7, v/v/v) and methylene chloride (100%) with flow rate at 1.0 mL/min and detection at 450 nm. All-trans-canthaxanthin was shown to be an appropriate internal standard for quantitation, with all-trans-β-carotene and its cis isomers present in largest amount (413.6 μg/g), followed by all-trans-violoxanthin and its cis isomers (209.5 μg/g), all-trans-lutein and its cis isomers (212.4 μg/g), all-trans-neoxanthin and its cis isomers (134.6 μg/g), antheraxanthin (16.5 μg/g), all-trans-β-cryptoxanthin and its cis isomers (5.8 μg/g), all-trans-zeaxanthin (3.6 μg/g) and neochrome (0.1 μg/g). For preparative chromatography, with a glass column containing 52 g of magnesium oxide-diatomaceous earth (1:3, w/w) as adsorbent, the carotenoid fraction was eluted with 300 mL of ethyl acetate with flow rate at 10 mL/min. Some more epoxides and cis isomers of carotenoids were generated during preparative column chromatography. Nevertheless, the carotenoids isolated from T. formosanum may be used as raw material for possible production of health food in the future.

  18. Micro-fabricated packed gas chromatography column based on laser etching technology.

    PubMed

    Sun, J H; Guan, F Y; Zhu, X F; Ning, Z W; Ma, T J; Liu, J H; Deng, T

    2016-01-15

    In this work, a micro packed gas chromatograph column integrated with a micro heater was fabricated by using laser etching technology (LET) for analyzing environmental gases. LET is a powerful tool to etch deep well-shaped channels on the glass wafer, and it is the most effective way to increase depth of channels. The fabricated packed GC column with a length of over 1.6m, to our best knowledge, which is the longest so far. In addition, the fabricated column with a rectangular cross section of 1.2mm (depth) × 0.6mm (width) has a large aspect ratio of 2:1. The results show that the fabricated packed column had a large sample capacity, achieved a separation efficiency of about 5800 plates/m and eluted highly symmetrical Gaussian peaks. PMID:26726935

  19. Micro-fabricated packed gas chromatography column based on laser etching technology.

    PubMed

    Sun, J H; Guan, F Y; Zhu, X F; Ning, Z W; Ma, T J; Liu, J H; Deng, T

    2016-01-15

    In this work, a micro packed gas chromatograph column integrated with a micro heater was fabricated by using laser etching technology (LET) for analyzing environmental gases. LET is a powerful tool to etch deep well-shaped channels on the glass wafer, and it is the most effective way to increase depth of channels. The fabricated packed GC column with a length of over 1.6m, to our best knowledge, which is the longest so far. In addition, the fabricated column with a rectangular cross section of 1.2mm (depth) × 0.6mm (width) has a large aspect ratio of 2:1. The results show that the fabricated packed column had a large sample capacity, achieved a separation efficiency of about 5800 plates/m and eluted highly symmetrical Gaussian peaks.

  20. New zwitterionic polymethacrylate monolithic columns for one- and two-dimensional microliquid chromatography.

    PubMed

    Jandera, Pavel; Staňková, Magda; Hájek, Tomáš

    2013-08-01

    We prepared 0.53 and 0.32 mm id monolithic microcolumns by in situ copolymerization of a zwitterionic sulfobetaine functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) and dioxyethylene dimetacrylate crosslinkers. The columns show a dual retention mechanism (hydrophilic-interaction mode) in acetonitrile-rich mobile phases and RP in highly aqueous mobile phases. The new 0.53 mm id columns provided excellent reproducibility, retention, and separation selectivity for phenolic acids and flavonoids. The new zwitterionic monolithic columns are highly orthogonal, with respect to alkyl silica stationary phases, not only in the hydrophilic-interaction mode but also in the RP mode. The optimized monolithic zwitterionic microcolumn of 0.53 mm id was employed in the first dimension, either in the aqueous normal-phase or in the RP mode, coupled with a short nonpolar core-shell column in the second dimension, for comprehensive 2D LC separations of phenolic and flavonoid compounds. When the 2D setup with the sulfobetaine-BIGDMA column was used for repeated sample analysis, with alternating gradients of decreasing (hydrophilic-interaction mode), and increasing (RP mode) concentration of acetonitrile on the sulfobetaine-BIGDMA column in the first dimension, useful complementary information on the sample could be obtained. PMID:23729220

  1. Polymethacrylate monolithic columns for hydrophilic interaction liquid chromatography prepared using a secondary surface polymerization.

    PubMed

    Currivan, Sinéad; Macak, Jan M; Jandera, Pavel

    2015-07-10

    Zwitterionic methacrylate based polymeric monolithic columns were prepared in two-step polymerizations, with reduced polymerization times. Characteristic properties such as hydrodynamic permeability, porosity, retention factors, and pore size distribution charts were used for column evaluation. A scaffold column was fabricated by polymerization of poly(lauryl methacrylate-co-tetraethyleneglycol dimethacrylate) and was used without further modification as a support for a poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine-co-bisphenol A glycerolate dimethacrylate) second monolith layer with zwitterionic functionality, for HILIC separations. An additional internal structure was formed by the second monolithic layer. The fabrication procedure was reproducible with RSD<5%. Field emission scanning electron microscopy has also been used to investigate column pore morphology, using a novel technique where the polymeric material is imaged directly, without coverage with a conducting film or particles. The new polar monolithic columns were used for HILIC separations of phenolic acids, flavones, nucleosides, and bases of nucleic acids, with similar efficiencies but different selectivities for zwitterionic methacrylate monolithic columns recently prepared by single step polymerization.

  2. Polymethacrylate monolithic columns for hydrophilic interaction liquid chromatography prepared using a secondary surface polymerization.

    PubMed

    Currivan, Sinéad; Macak, Jan M; Jandera, Pavel

    2015-07-10

    Zwitterionic methacrylate based polymeric monolithic columns were prepared in two-step polymerizations, with reduced polymerization times. Characteristic properties such as hydrodynamic permeability, porosity, retention factors, and pore size distribution charts were used for column evaluation. A scaffold column was fabricated by polymerization of poly(lauryl methacrylate-co-tetraethyleneglycol dimethacrylate) and was used without further modification as a support for a poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine-co-bisphenol A glycerolate dimethacrylate) second monolith layer with zwitterionic functionality, for HILIC separations. An additional internal structure was formed by the second monolithic layer. The fabrication procedure was reproducible with RSD<5%. Field emission scanning electron microscopy has also been used to investigate column pore morphology, using a novel technique where the polymeric material is imaged directly, without coverage with a conducting film or particles. The new polar monolithic columns were used for HILIC separations of phenolic acids, flavones, nucleosides, and bases of nucleic acids, with similar efficiencies but different selectivities for zwitterionic methacrylate monolithic columns recently prepared by single step polymerization. PMID:26022313

  3. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    PubMed

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

  4. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    PubMed

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants. PMID:18307162

  5. Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study.

    PubMed

    Stroka, J; Anklam, E; Jörissen, U; Gilbert, J

    2000-01-01

    A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 and total aflatoxins at European regulatory limits. The test portion is extracted with methanol-water (8 + 2) for dried figs and paprika, and with methanol-water (8 + 2) plus hexane (or cyclohexane) for peanut butter and pistachios. The sample extract is filtered, diluted with phosphate buffer saline, and applied to an immunoaffinity column. The column is washed with water and the aflatoxins are eluted with methanol. Aflatoxins are quantitated by reversed-phase LC with post-column derivatization (PCD) involving bromination. PCD is achieved with either an electrochemical cell (Kobra cell) and addition of bromide to the mobile phase or pyridinium hydrobromide perbromide. Determination is by fluorescence. Peanut butter, pistachio paste, dried fig paste, and paprika powder samples, both naturally contaminated with aflatoxins and containing added aflatoxins, were sent to 16 collaborators in 16 European countries. Test portions of samples were spiked at levels of 2.4 and 9.6 ng/g for total aflatoxins which included 1.0 and 4.0 ng/g aflatoxin B1, respectively. Recoveries for total aflatoxins ranged from 71 to 92% with corresponding recoveries for aflatoxin B1 of 82 to 109%. Based on results for spiked samples (blind duplicates at 2 levels) as well as naturally contaminated samples (blind duplicates at 4 levels, including blank), the relative standard deviation for repeatability ranged from 4.6 to 23.3% for total aflatoxins and from 3.1 to 20.0% for aflatoxin B1. The relative standard deviation for reproducibility ranged from 14.1 to 34.2% for total aflatoxins, and from 9.1 to 32.2% for aflatoxin B1. The method showed acceptable within-laboratory and between-laboratory precision for all 4 matrixes, as evidenced by HORRAT values <1, at the low levels of determination for both total aflatoxins and

  6. Purification of phosphinothricin acetyltransferase using Reactive brown 10 affinity in a single chromatography step.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2013-08-01

    The expression of phosphinothricin N-acetyltransferase (PAT) protein in transgenic plants confers tolerance to the herbicide glufosinate. To enable the characterization of PAT protein expressed in plants, it is necessary to obtain high purity PAT protein from the transgenic grain. Because transgenically expressed proteins are typical present at very low levels (i.e. 0.1-50 μg protein/g grain), a highly specific and efficient purification protocol is required to purify them. Based on the physicochemical properties of PAT, we developed a novel purification method that is simple, time-saving, inexpensive and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein was purified to homogeneity from cottonseed with high recovery efficiency. As expected, the Reactive brown 10-produced PAT was enzymatically active. Other applications of the method on protein expression and purification, and development of PAT enzymatic inhibitors were also discussed. PMID:23748142

  7. [Determination of the interaction kinetics between meloxicam and β-cyclodextrin using the quantitative high-performance affinity chromatography coupled with mass spectrometry].

    PubMed

    Wang, Cai-fen; Li, Zhuo; Wang, Xiao-bo; Li, Hai-yan; Zhang, Ji-wen; Sun, Li-xin

    2015-09-01

    The association rate constant and dissociation rate constant are important parameters of the drug-cyclodextrin supermolecule systems, which determine the dissociation of drugs from the complex and the further in vivo absorption of drugs. However, the current studies of drug-cyclodextrin interactions mostly focus on the thermodynamic parameter of equilibrium constants (K). In this paper, a method based on quantitative high performance affinity chromatography coupled with mass spectrometry was developed to determine the apparent dissociation rate constant (k(off,app)) of drug-cyclodextrin supermolecule systems. This method was employed to measure the k(off,app) of meloxicam and acetaminophen. Firstly, chromatographic peaks of drugs and non-retained solute (uracil) on β-cyclodextrin column at different flow rates were acquired, and the retention time and variance values were obtained via the fitting the peaks. Then, the plate heights of drugs (H(R)) and uracil (H(M,C)) were calculated. The plate height of theoretical non-retained solute (H(M,T)) was calculated based on the differences of diffusion coefficient and the stagnant mobile phase mass transfer between drugs and uracil. Finally, the k(off,app) was calculated from the slope of the regression equation between (H(R)-H(M,T)) and uk/(1+k)2, (0.13 ± 0.00) s(-1) and (4.83 ± 0.10) s(-1) for meloxicam and acetaminophen (control drug), respectively. In addition, the apparent association rate constant (k(on,app)) was also calculated through the product of K (12.53 L x mol(-1)) and k(off,app). In summary, it has been proved that the method established in our study was simple, efficiently fast and reproducible for investigation on the kinetics of drug-cyclodextrin interactions. PMID:26757555

  8. Determination of a highly selective mixed-affinity sigma receptor ligand, in rat plasma by ultra performance liquid chromatography mass spectrometry and its application to a pharmacokinetic study

    PubMed Central

    Jamalapuram, Seshulatha; Vuppala, Pradeep K.; Mesangeau, Christophe; McCurdy, Christopher R.; Avery, Bonnie A.

    2014-01-01

    A selective, rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC/MS) method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand, CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d] thiazole-2(3H)-thione), in rat plasma. CM156 and the internal standard (aripiprazole) were extracted from plasma samples by a single step liquid–liquid extraction using chloroform. The analysis was carried out on an ACQUITY UPLCTM BEH HILIC column (1.7 µm, 2.1 mm × 50 mm) with isocratic elution at flow rate of 0.2 mL/min using 10 mM ammonium formate in 0.1% formic acid and acetonitrile (10:90) as the mobile phase. The detection of the analyte was performed on a mass spectrometer operated in selected ion recording (SIR) mode with positive electrospray ionization (ESI). The validated analytical method resulted in a run time of 4 min and the retention times observed were 2.6 ± 0.1 and 2.1 ± 0.1 min for CM156 and the IS, respectively. The calibration curve exhibited excellent linearity over a concentration range of 5–4000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra- and inter-day precision values were below 15% and accuracy ranged from −6.5% to 5.0%. The mean recovery of CM156 from plasma was 96.8%. The validated method was applied to a pilot intravenous pharmacokinetic study in rats. PMID:22406103

  9. sup 32 P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase

    SciTech Connect

    Reddy, M.V.; Bleicher, W.T.; Blackburn, G.R. )

    1991-04-01

    Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive {sup 32}P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO{sub 4}). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO{sub 4}-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO{sub 4} selectively forms cis-Tg adducts. With OsO{sub 4}-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO{sub 4}-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.

  10. Comparison of chromatographic band profiles obtained under microwave irradiated and non-irradiated reversed-phase liquid chromatography column

    SciTech Connect

    Galinada, Wilmer; Guiochon, Georges A

    2005-08-01

    The possible influence of the application of microwave energy to a reversed-phase liquid chromatography column on the mass transfer kinetics and the thermodynamics of equilibrium between mobile and stationary phases was examined. Chromatograms of propylbenzene and phenol were recorded under the same experimental conditions, on the same column, successively irradiated and not. The effect of microwave irradiation on the mass transfer kinetics was determined by measuring the second moment of small pulses of propylbenzene in a 70:30 (v/v) solution of methanol in water and microwave outputs of 15 and 30 W. The effect of microwave irradiation on the equilibrium thermodynamics was determined by measuring the elution time of breakthrough curves of phenol at high concentrations in a 20:80 (v/v) solution of methanol and water and microwave outputs of 15, 50, and 150 W. A qualitative comparison of the profiles of the propylbenzene peaks obtained with and without irradiation suggests that this irradiation affects significantly the peak shapes. However, a qualitative comparison of the profiles of the breakthrough curves of phenol obtained with and without irradiation suggests that this irradiation has no significant effect on their shapes. The peak sharpening observed may be due to an increase in the diffusivity, resulting from the dielectric polarization under microwave irradiation. This effect is directly related to an increase of the rate of mass transfers in the column. In contrast, the similarity of the overloaded band profiles at high concentrations suggests that the equilibrium thermodynamics is unaffected by microwave irradiation. This may be explained by the transparence of the stationary phase to microwaves at 2.45 GHz. The column temperature was measured at the column outlet under irradiation powers of 15, 30, 50, and 150 W. It increases with increasing power, the corresponding effluent temperatures being 25 {+-} 1, 30 {+-} 1, 35 {+-} 1, and 45 {+-} 1 C, respectively.

  11. Comparison of chromatographic band profiles obtained under microwave irradiated and non-irradiated reversed-phase liquid chromatography column.

    PubMed

    Galinada, Wilmer A; Guiochon, Georges

    2005-10-28

    The possible influence of the application of microwave energy to a reversed-phase liquid chromatography column on the mass transfer kinetics and the thermodynamics of equilibrium between mobile and stationary phases was examined. Chromatograms of propylbenzene and phenol were recorded under the same experimental conditions, on the same column, successively irradiated and not. The effect of microwave irradiation on the mass transfer kinetics was determined by measuring the second moment of small pulses of propylbenzene in a 70:30 (v/v) solution of methanol in water and microwave outputs of 15 and 30 W. The effect of microwave irradiation on the equilibrium thermodynamics was determined by measuring the elution time of breakthrough curves of phenol at high concentrations in a 20:80 (v/v) solution of methanol and water and microwave outputs of 15, 50, and 150 W. A qualitative comparison of the profiles of the propylbenzene peaks obtained with and without irradiation suggests that this irradiation affects significantly the peak shapes. However, a qualitative comparison of the profiles of the breakthrough curves of phenol obtained with and without irradiation suggests that this irradiation has no significant effect on their shapes. The peak sharpening observed may be due to an increase in the diffusivity, resulting from the dielectric polarization under microwave irradiation. This effect is directly related to an increase of the rate of mass transfers in the column. In contrast, the similarity of the overloaded band profiles at high concentrations suggests that the equilibrium thermodynamics is unaffected by microwave irradiation. This may be explained by the transparence of the stationary phase to microwaves at 2.45 GHz. The column temperature was measured at the column outlet under irradiation powers of 15, 30, 50, and 150 W. It increases with increasing power, the corresponding effluent temperatures being 25+/-1, 30+/-1, 35+/-1, and 45+/-1 degrees C, respectively.

  12. Extreme scale-down approaches for rapid chromatography column design and scale-up during bioprocess development.

    PubMed

    Chhatre, Sunil

    2013-01-01

    Chromatography is a ubiquitous protein purification step owing to its unparalleled ability to recover and purify molecules from highly complex industrial feedstocks. Traditionally, column development has been driven by a combination of prior experience and empirical studies in order to make the best choices for design variables. Economic constraints now demand that companies engage with a more systematic exploration of a chromatographic design space. To deliver this capability using purely conventional laboratory columns, however, would require considerable resources to identify practical and economical operating protocols. Hence, recently there has been increased use of extremely small-scale devices that gather data quickly and with minimal feed requirements. Such information can be obtained either during early development for screening and trend-finding purposes or later for more accurate scale-up prediction. This chapter describes some of the key drivers for these small-scale studies and the different types of extreme scale-down chromatography formats that exist and illustrates their use through published case studies. Since extreme scale-down experimentation is linked to fundamental mechanistic engineering approaches as well, the utility of these in delivering process understanding is also highlighted.

  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. Vortex Counter-Current Chromatography: Performance of a New Preparative Column

    PubMed Central

    Ito, Yoichiro; Clary, Robert; Witten, Jacob J.; Zeng, Yun

    2012-01-01

    A New preparative column for the vortex counter-current chromatograph was fabricated by making many (966) cylindrical separation units to a high-dendity polyethylene disk and thenthreading them with 6-40 taps. The resulting column had a total capacity of 364 mL. The performance of this vortex column was examined with three different two-phase solvent systems each using a set of suitable test samples: hexane-ethyl acetate-methanol-0.1 M hydrochloric acid (1:1:1:1, v/v) for separation of DNP-amino acids; 1-butanol-acetic acid-water (4:1:5, v/v) for separation of dipeptides; and hexane-acetonitrile-water (20:15:2, v/v) for separation of Sudan dyes. Most of the separations show high partition efficiency of over a thousand theoretical plates, as expected based on the results previously obtained in preliminary separations with a small column. Overall, the results of the present study suggest that further improvement of the partition efficiency can be obtained by the modifying column configuration. PMID:23503931

  15. Conformational plasticity of IgG during protein A affinity chromatography.

    PubMed

    Gagnon, Pete; Nian, Rui

    2016-02-12

    Single step elution of a protein A column with 100mM acetate pH 3.5 produced a curvilinear gradient with pH dropping steeply at first then more gradually as it approached endpoint. IgG with a native hydrodynamic diameter of 11.5 nm began to elute at pH 6.0 with a size of 9.4 nm. IgG size continued to decrease across the peak, reaching a minimum of 2.2 nm at pH 3.9. Secondary structure of early eluting IgG was only mildly affected but later eluting fractions became increasingly non-native with the 2.2 nm population exhibiting the highest proportion of β-sheet and lowest random coil of all conformations. Size reduction and structural change of IgG through this portion of the elution peak were attributed dominantly to a pre-existing tendency of highly concentrated IgG to adopt reduced size conformations at low pH and conductivity, facilitated by the known conformational relaxation of IgG by its interaction with protein A. IgG size increased to 10.4 nm as elution pH approached 3.5 across the tailing fractions. Major loss of β-sheet and increase of α-helix and random coil were observed in parallel. Late elution of this population was attributed to it being eluted from interactions with 2 distinct protein A domains, one bound to each side of the Fc region, creating a higher dissociation constant than single-site Fc-protein A interactions, and requiring more severely disruptive conditions for elution. The high degree of conformational disruption was attributed to simultaneous interaction of both heavy chains with protein A. PMID:26805601

  16. Examination of Glycan Profiles from IgG-Depleted Human Immunoglobulins Facilitated by Microscale Affinity Chromatography

    PubMed Central

    Svoboda, Martin; Mann, Benjamin F.; Goetz, John A.; Novotny, Milos V.

    2012-01-01

    Among the most important proteins involved in the disease and healing processes are the immunoglobulins (Igs). Although many of the Igs have been studied through proteomics, aside from IgG, immunoglobulin carbohydrates have not been extensively characterized in different states of health. It seems valuable to develop techniques that permit us to understand changes in the structures and abundances of Ig glycans in the context of disease onset and progression. We have devised a strategy for characterization of the glycans for the Ig classes other than IgG (i.e. A, D, E, and M) that contain kappa light chains, while using only a few microliters of biological material. First, we designed a microcolumn containing the recombinant Protein L that was immobilized on macroporous silica particles. A similarly designed Protein G microcolumn was utilized to first perform an on-line depletion of the IgG from the sample, human blood serum, and thereby facilitate enrichment of the other Igs. While only 3 μL of serum were used in these analyses, we were able to recover a significantly-enriched fraction of non-IgG immunoglobulins. The enrichment properties of the Protein L column were characterized using a highly sensitive label-free quantitative proteomics LC-MS/MS approach, and the glycomic profiles of enriched immunoglobulins were measured by MALDI-TOF-MS. As a proof-of-principle, a comparative study was conducted using blood serum from a small group of lung cancer patients and a group of age-matched cancer-free individuals to demonstrate that the method is suitable for investigation of glycosylation changes in disease. The results were in agreement with a glycomic investigation of whole blood serum from a much larger lung cancer cohort. PMID:22360417

  17. Comparative study of glycated hemoglobin by ion exchange chromatography and affinity binding nycocard reader in type 2 diabetes mellitus.

    PubMed

    Gautam, N; Dubey, R K; Jayan, A; Nepaune, Y; Padmavathi, P; Chaudhary, S; Jha, S K; Sinha, A K

    2014-12-01

    The aim of this study was to compare the level of glycated hemoglobin (HbA1c) in type 2 Diabetes Mellitus (DM) patients by two different methods namely Ion Exchange Chromatography and Affinity Binding Nycocard Reader. This is a cross-sectional study conducted on confirmed type 2 diabetes mellitus patients (n = 100) who visited Out Patients Department of the Universal College of Medical Sciences Teaching hospital, Bhairahawa, Nepal from November 2012 to March 2013. The diagnosis of diabetes mellitus was done on the basis of their fasting (164.46 ± 45.33 mg/dl) and random (187.93 ± 78.02 mg/dl) serum glucose level along with clinical history highly suggestive of type 2 DM. The HbA1c values of (7.8 ± 1.9%) and (8.0 ± 2.2%) were found in DM patients as estimated by those two different methods respectively. The highest frequency was observed in HbA1c > 8.0% indicating maximum cases were under very poor glycemic control. However, there were no significant differences observed in HbA1c value showing both methods are comparable in nature and can be used in lab for ease of estimation. The significant raised in HbA1c indicates complications associated with DM and monitoring of therapy become hard for those patients. Despite having standard reference method for HbA1c determination, the availability of report at the time of the patient visit can be made easy by using Nycocard Reader and Ion Exchange Chromatography techniques without any delay in communicating glycemic control, clinical decision-making and changes in treatment regimen.

  18. Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

    PubMed Central

    Gounaris, A D; Brown, M A; Barrett, A J

    1984-01-01

    Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated. Images Fig. 2. Fig. 3. Fig. 4. PMID:6548132

  19. Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

    PubMed Central

    Watson, D H; Wootton, J C

    1977-01-01

    The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure

  20. Problems in the size exclusion chromatography of poly( N-isopropylacrylamide) on styragel columns

    NASA Astrophysics Data System (ADS)

    Estrin, Ya. I.; Perepelitsina, E. O.; Grishchuk, A. A.

    2016-07-01

    The molecular weights of poly( N-isopropylacrylamide) (PNIPA), calculated according to polystyrene calibration standards upon the elution of THF on styragel columns, appear to be much lower than their actual values determined using independent approaches. This is likely due to interactions between the nitrogen-containing units of PNIPA polymer chains and the sorbent, so the polymer is eluted in the mode intermediate between exclusion and critical. An effective exclusion mode during the elution of PNIPA on a styragel column can be achieved by using an eluent more polar than tetrahydrofuran (particularly, 1-methylpyrrolidone).