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Chromatography System (chromatography + system)
Kinds of Chromatography System Selected AbstractsMicroaffinity purification of proteins based on photolytic elution: Toward an efficient microbead affinity chromatography on a chipELECTROPHORESIS, Issue 3 2005Woo-Jae Chung Abstract A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore® beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated. [source] Effect of Conductivity, pH, and Elution Buffer Salinity on Glycomacropeptide Recovery from Whey Using Anion Exchange ChromatographyJOURNAL OF FOOD SCIENCE, Issue 4 2005Hatice N. Tek ABSTRACT: The objective of this study was to investigate the effect of whey conductivity, pH, and the salt concentration of the elution buffer on glycomacropeptide recovery and its extent of contamination using anion exchange chromatography. Glycomacropeptide was isolated from Mozzarella whey. Samples were analyzed for glycomacropeptide and contaminating whey proteins. Mass balances and percent recoveries were calculated from these data. Glycomacropeptide recovery increased substantially with decreasing conductivity and increasing pH of the whey feed stream. Increasing the pH, but not increasing the conductivity, increased contamination of the glycomacropeptide by primarily beta-lactoglobulin. Salt concentration of at least 0.1 M was required for complete elution of bound glycomacropeptide. These data define conditions needed for glycomacropeptide recovery by a process chromatography system that uses food-grade buffers, operates at industrially relevant flow rates, and achieves up to 98% recovery. [source] In vitro assessment of cytochrome P450 inhibition: Strategies for increasing LC/MS-based assay throughput using a one-point IC50 method and multiplexing high-performance liquid chromatographyJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2007Tong Lin Abstract A fast and robust LC/MS-based cytochrome P450 (CYP) inhibition assay, using human liver microsomes, has been fully developed and validated for the major human liver CYPs. Probe substrates were phenacetin, diclofenac, S-mephenytoin, and dextromethorphan for CYP1A2, CYP2C9, CYP2C19, and CYP2D6, respectively. Midazolam and testosterone were chosen for CYP3A4. Furafylline, sulfaphenazole, tranylcypromine, quinidine, and ketoconazole were identified as positive control inhibitors for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. To increase the throughput of the assay, a one-point method was developed, using data from CYP inhibition assays conducted at one concentration (i.e., 10 µM), to estimate the drug concentration at which the metabolism of the CYP probe substrate was reduced by 50% (IC50). The IC50 values from the one-point assay were validated by correlating the results with IC50 values that were obtained with a traditional eight-point concentration,response curve. Good correlation was achieved with the slopes of the trendlines between 0.95 and 1.02 and with R2 between 0.77 and 1.0. Throughput was increased twofold by using a Cohesive multiplexing high-performance liquid chromatography system. The one-point IC50 estimate is useful for initial compound screening, while the full concentration,response IC50 method provides detailed CYP inhibition data for later stages of drug development. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 2485,2493, 2007 [source] Enrichment of peptides from plasma for peptidome analysis using multiwalled carbon nanotubesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6 2007Xin Li Abstract Human plasma contains a complex matrix of proteolytically derived peptides (plasma peptidome) that may provide a correlate of biological events occurring in the entire organism. Analyzing these peptides from a small amount of serum/plasma is difficult due to the complexity of the sample and the low levels of these peptides. Here, we describe a novel peptidome analysis approach using multiwalled carbon nanotubes (MWCNTs) as an alternative adsorbent to capture endogenous peptides from human plasma. Harvested peptides were analyzed by using liquid chromatography-mass spectrometry as a means of detecting and assessing the adsorbed molecules. The improved sensitivity and resolution obtained by using liquid chromatography-mass spectrometry allowed detection of 2521 peptide features (m/z 300,1800 range) in about 50 ,L of plasma. 374 unique peptides were identified with high confidence by two-dimensional liquid chromatography system coupled to a nano-spray ionization linear ion trap-mass spectrometer. High recovery of BSA digest peptides enriched with MWCNTs, in both standard buffer and high abundance protein solution, was observed. Comparative studies showed that MWCNTs were superior to C18 and C8 for the capture of the smaller peptides. This approach could hold promise of routine plasma peptidome analysis. [source] Accumulation of Hemoglobin-Associated Acetaldehyde With Habitual Alcohol Drinking in the Atypical ALDH GenotypeALCOHOLISM, Issue 1 2000Tatsuya Takeshita Background: Those with the atypical genotypes of low Km aldehyde dehydrogenase (ALDH2) have high blood concentrations of free acetaldehyde, an active metabolite of ethanol, after drinking alcohol. In the present study, we measured acetaldehyde reversibly bound to hemoglobin (HbAA) in Japanese male workers. Methods: One hundred and sixty Japanese male workers in one plant participated with informed consent. The subjects were genotyped for the ALDH2 polymorphism by polymerase chain reaction method. HbAA levels were measured using a high performance liquid chromatography system with a fluorescence detector. For the study in which we examined accumulation of HbAA, eight Asian male volunteers participated with informed consent. Results: Although HbAA levels were significantly correlated with recent alcohol consumption in both typical (ALDH2*1/*1) and atypical (ALDH2*1/*2)genotypes, the slope in ALDH2*1/*2 was significantly steeper than that in ALDH2*1/*1. Multiple regression analysis on relevant factors for HbAA revealed that not only recent but also daily alcohol consumption increased HbAA levels in those with the ALDH2*1/*2 genotype, which suggests that HbAA accumulates with habitual drinking. We measured HbAA levels before, during, and after alcohol consumption,one drink (0.4 ml/kg) per day,for 7 consecutive days in male volunteers. During the drinking period, HbAA lincarly increased in ALDH2*1/*2 (n= 4) but not in ALDH2*1/*1 (n= 4). After reaching peak levels (+76.1 nmol/g hemoglobin) following the seventh drink, HbAA levels gradually decreased but were significantly higher for 3 days after drinking was discontinued. Conclusions: We demonstrated that HbAA levels accumulate with habitual alcohol drinking in the atypical ALDH2 genotype. HbAA was shown to be a good biomarker for increased internal exposure levels to acetaldehyde. [source] Determination and toxicokinetics comparison of ketamine and S(+)-ketamine in dog plasma by HPLC-MS methodBIOMEDICAL CHROMATOGRAPHY, Issue 3 2010Yu-xin Sheng Abstract ;A simple and reproducible method was developed for the quantification of ketamine and S(+)-ketamine in dog plasma using a high-performance liquid chromatography system coupled to a positive ion electrospray mass spectrometric analysis. Solid-phase extraction was used for extracting analytes from dog plasma samples. The analytes were separated on a Zorbax SB C18 column (100 × 2.1 mm, 3.5 ,m) with acetonitrile,formate buffer (10 mM ammonium formate and 0.3% formic acid) (17 : 83, v/v) as mobile phase at a flow-rate of 0.2 mL/min. Detection was operated under selected ion monitoring mode. [M + H]+ at m/z 238 for ketamine and S(+)-ketamine and [M + H]+ at m/z 180 for phenacetin (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration range 51.6,2580 ng/mL. The intra- and inter-day precisions (RSD %) were within 11.3% and the assay accuracies ranged from 80.0 to 101.4%. Their average recoveries were greater than 91.1% at all test concentrations. The analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the toxicokinetics study and comparison of ketamine and S (+)-ketamine following intravenous administration to dogs. Copyright © 2009 John Wiley & Sons, Ltd. [source] Preparation of a super-long two column chromatography system and its application in separating glycosylated puerarinBIOMEDICAL CHROMATOGRAPHY, Issue 12 2009Shouchuang Zhu Abstract Separation of Puerarin-7- O -glucoside from its precursor, puerarin, using a common chromatography column packed with AB-8 macroporous resin was unsuccessful. Therefore, in this study a 8,m super-long flexible reinforced PVC column was externally added to the common column in order to improve the chromatography efficiency by increasing the number of theoretical plates. Both the PVC and common columns were separately packed with AB-8 macroporous resin slurry. The packed PVC column was coiled after washing and stored until use. The microbial transformation mixture with puerarin-7- O -glucoside and puerarin (250,mL) was loaded onto the common column, followed by washing with 2000,mL H2O. After attaching the coiled external PVC column to the common column, a linear gradient of 10,30% ethanol was applied to elute the target compound. Two peaks appeared: peak I contained puerarin-7- O -glucoside at 97.9% purity and 88.1% recovery rate, and peak II was puerarin at 98.7% purity and 87.0% recovery rate. The use of the coiled external flexible reinforced PVC column avoided spatial restriction for long columns, which made it much more convenient for column packing and chromatography operations. Furthermore, this method eliminated the resin blockage problem caused by stationary water pressure in a rigid vertical long column. Using an external super-long column, the PVC tube was connected with the common column only during elution, which avoided delay in time period during sample loading and column washes associated with the use of long external columns. Copyright © 2009 John Wiley & Sons, Ltd. [source] High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolitesBIOMEDICAL CHROMATOGRAPHY, Issue 1 2009Shuang Fu Abstract Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones,desmethoxyyangonin, methysticin and kavain,in rat liver microsomes using diazepam as an internal standard; liquid,liquid extraction was used for sample preparation and analysis was performed on a Shimadzu® 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna® C18 column (150 × 2.00 mm, 3 µm) at 40°C. The limit of quantitation was 0.1 µg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1,10 µg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites. Copyright © 2008 John Wiley & Sons, Ltd. [source] Automated analysis of fluvoxamine in rat plasma using a column-switching system and ion-pair high-performance liquid chromatographyBIOMEDICAL CHROMATOGRAPHY, Issue 12 2008Shicheng Liu Abstract We have established a robust, fully automated analytical method for the analysis of fluvoxamine in rat plasma using a column-switching ion-pair high-performance chromatography system. The plasma sample was injected onto a precolumn packed with Shim-pack MAYI-ODS (50 µm), where the drug was automatically purified and enriched by on-line solid-phase extraction. After elution of the plasma proteins, the analyte was back-flushed from the precolumn and then separated isocratically on a reversed-phase C18 column (L-column ODS) with a mobile phase (acetonitrile,0.1% phosphoric acid, 36:64, v/v) containing 2 mm sodium 1-octanesulfonate. The analyte was monitored by a UV detector at a wavelength of 254 nm. The calibration line for fluvoxamine showed good linearity in the range of 5,5000 ng/mL (r > 0.999) with the limit of quantification of 5 ng/mL (RSD = 6.51%). Accuracy ranged from ,2.94 to 4.82%, and the within- and between-day precision of the assay was better than 8% across the calibration range. The analytical sensitivity and accuracy of this assay is suitable for characterization of the pharmacokinetics of orally-administered fluvoxamine in rats. Copyright © 2008 John Wiley & Sons, Ltd. [source] Improved RP-HPLC determination of quinine in plasma and whole blood stored on filter paperBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 9 2000J.A. Kolawole Abstract Analysis of quinine in plasma and whole blood samples dried on filter paper is described. Sample preparation involves liquid extraction of plasma and whole blood from the filter paper and subsequent solid-phase extraction using C8 Bond Elut cartridges. A reverse-phase liquid chromatography system with UV detection and fluorescence detection was used. The analytical characteristics of the method are reported, with a quantification limit of 0.1 µg mL,1 and within an assay coefficient of variation of 5.6,8.4% in plasma and 6.5,12% in whole blood. Representative chromatograms are shown as a function of time for samples from human subjects after ingestion of a single 400-mg dose of quinine sulphate. Quinidine, dihydroquinine and metabolites are well separated from quinine with a resolution of above 1 (Rs>1). Copyright © 2000 John Wiley & Sons, Ltd. [source] Inexpensive and Generic Affinity Purification of Recombinant Proteins Using a Family 2a CBM Fusion TagBIOTECHNOLOGY PROGRESS, Issue 5 2004Beatriz Rodriguez The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellullose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 ± 3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein. [source] An Integrated Process for Mammalian Cell Perfusion Cultivation and Product Purification Using a Dynamic FilterBIOTECHNOLOGY PROGRESS, Issue 4 2002Leda R. Castilho In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process. A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments. In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations. The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL,1, whereby cell viability remained above 90% and no problems of sterility were experienced. In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands. An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system. The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles. After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out. No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells. [source] Chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether for enantiomer separation of amino compounds using a normal mobile phaseCHIRALITY, Issue 3 2005Keiji Hirose Abstract In order to apply the excellent chiral recognition ability of chiral pseudo-18-crown-6 ethers that we developed to chiral separation, we prepared a chiral stationary phase (CSP) by immobilizing a chiral pseudo-18-crown-6-type host on 3-aminopropyl silica gel. A chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. A liquid chromatography system using this CSP combined with the detection by mass spectrometry was used for enantiomer separation of amino compounds. A normal mobile phase can be used on this CSP as opposed to conventional dynamic coating-type CSPs. Enantiomers of 18 common natural amino acids were efficiently separated. The chiral separation observed for amino acid methyl esters, amino alcohols, and lipophilic amines was fair using this HPLC system. In view of the correlation between the enantiomer selectivity observed in chromatography and the complexion in solution, the chiral recognition in host,guest interactions might contribute to this enantiomer separation. Chirality 17:142,148, 2005. © 2005 Wiley-Liss, Inc. [source] Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at-line tool for making pooling decisions for process chromatographyBIOTECHNOLOGY PROGRESS, Issue 5 2009Anurag S. Rathore Abstract Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online-high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real-time decisions for column pooling based on product quality attributes (Rathore et al., 2008a,b). In this article, we review an at-line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] High-throughput analysis of in vitro cytochrome p450 inhibition samples using mass spectrometry coupled with an integrated liquid chromatography/autosampler systemRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 8 2010Ann Brown First page of article [source] | ||||||||||||||||