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Interaction Chromatography (interaction + chromatography)
Kinds of Interaction Chromatography Selected AbstractsSynthesis of Sugar-Based Silica Gels by Copper-Catalysed Azide,Alkyne Cycloaddition via a Single-Step Azido-Activated Silica Intermediate and the Use of the Gels in Hydrophilic Interaction ChromatographyCHEMISTRY - A EUROPEAN JOURNAL, Issue 19 2010Lisa Moni Dr. Abstract Novel sugar-based silica gels were prepared by exploiting the copper-catalysed azide,alkyne cycloaddition (CuAAC) of two different sugar alkynes, namely, ethynyl C -galactoside 1 and propargyl O -lactoside 2, with new single-step azido-activated silica gels. The fully characterised stationary phases were generally used for hydrophilic interaction chromatography (HILIC), with particular application in the stereoselective separation of monosaccharides. Dynamic HILIC (DHILIC) experiments were performed to evaluate the influence of mutarotation on the chromatographic peak shapes of two interconverting sugar anomers. The potential of such materials was shown in the separation of other highly polar compounds, including amino acids and flavonoids. [source] Retinol binding protein isolated from acute renal failure patients inhibits polymorphonuclear leucocyte functionsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2004G. Cohen Abstract Background, Protein factors accumulating in sera of patients with end-stage renal disease (ESRD) that interfere with the nonspecific immune response by inhibiting essential functions of polymorphonuclear leucocytes (PMNLs) have previously been described. No such factor has been isolated from acute renal failure (ARF) patients to date. Materials and methods, Using a three-step chromatographic procedure involving ion exchange, size exclusion and hydrophobic interaction chromatography we purified the apo- and holo-form of retinol binding protein (RBP) from high-flux dialyser (polyacrylonitrile; AN69) ultrafiltrates of patients with ARF. Their effect on the chemotaxis of PMNLs isolated from healthy donors was determined by the under-agarose method. Whole-blood assays applying flow cytometry were used to assess phagocytosis and the oxidative metabolism of PMNLs. Apoptosis was assessed by determining the DNA content using propidium iodide. Results, Isolated apo- and holo-forms of RBP were truncated on their C-terminus as determined by mass spectrometry. All isolates significantly inhibited the chemotactic movement of PMNLs obtained from healthy donors and the PMNL oxidative metabolism stimulated by E. coli. These effects were concentration dependent. Retinol binding protein had no influence on the PMNL oxidative metabolism stimulated by PMA and on PMNL phagocytosis. Commercially available RBP isolated from urine influenced PMNL functions in the same way. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 significantly attenuated the phagocytosis-induced respiratory burst and RBP did not lead to a further decrease. Polymorphonuclear leucocyte apoptosis was significantly inhibited by RBP. Conclusions, The apo- and holo-forms of RBP isolated from the ultrafiltrate of ARF patients inhibit PMNL chemotaxis, oxidative metabolism and apoptosis. Therefore, RBP may be considered a uraemic toxin contributing to a disturbed immune defence. [source] Towards a platform for the metabonomic profiling of different strains of Drosophila melanogaster using liquid chromatography,Fourier transform mass spectrometryFEBS JOURNAL, Issue 22 2009Muhammad A. Kamleh A platform based on hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry was developed in order to carry out metabonomics of Drosophila melanogaster strains. The method was able to detect , 230 metabolites, mainly in the positive ion mode, after checking to eliminate false positives caused by isotope peaks, adducts and fragment ions. Two wild-type strains, Canton S and Oregon R, were studied, plus two mutant strains, Maroon Like and Chocolate. In order to observe the differential expression of metabolites, liquid chromatography-mass spectrometry analyses of the different strains were compared using sieve 1.2 software to extract metabolic differences. The output from sieve was searched against a metabolite database using an Excel-based macro written in-house. Metabolic differences were observed between the wild-type strains, and also between both Chocolate and Maroon Like compared with Oregon R. It was established that a metabonomic approach could produce results leading to the generation of new hypotheses. In addition, the structure of a new class of lipid with a histidine head group, found in all of the strains of flies, but lower in Maroon Like, was elucidated. [source] Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthodermaFEBS JOURNAL, Issue 3 2007Magdalena Kujawa We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose,methanol,choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9- S -cysteinyl, 8-,- N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are d -glucose, d -galactose, l -arabinose, and d -xylose. As shown by in situ NMR analysis, d -glucose and d -galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (kcat/Km). The enzyme may play a role in lignocellulose degradation. [source] Investigation into the effect of detergents on disinfectant susceptibility of attached Escherichia coli and Listeria monocytogenesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2008J.T. Walton Abstract Aims:, Investigate the effect of detergent treatment on susceptibility of attached Escherichia coli and Listeria monocytogenes to subsequent disinfectant treatment. Methods and Results:, Plate counts show that E. coli attached to stainless steel surfaces became significantly more susceptible to benzalkonium chloride (BAC) after treatment with sodium alkyl sulfate (SAS) and fatty alcohol ethoxylate (FAE). No change in susceptibility was observed with Sodium dodecyl sulfate (SDS). L. monocytogenes became significantly less susceptible to BAC after treatment with SAS and SDS yet no change in susceptibility was observed with FAE. Flow cytometry using the fluoresceine propidium iodide revealed significant increases in cell membrane permeability of both organisms by SAS and FAE, although the effect was much greater in E. coli. No change was observed with SDS. Hydrophobic interaction chromatography showed that both organisms became less hydrophobic following treatment with SAS and SDS but FAE had no effect. Conclusions:, In E. coli, detergents that increase susceptibility to BAC increase membrane permeability. In L. monocytogenes, detergents that reduce susceptibility to BAC lower cell surface hydrophobicity. Significance and Impact of the Study:, Detergents can influence the sensitivity of pathogenic food borne micro-organisms to BAC. [source] Physicochemical properties of Shiga toxigenic Escherichia coliJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2005L. Rivas Abstract Aims:, To investigate the physicochemical surface properties, such as cellular surface charge, hydrophobicity and electron donor/acceptor potential of a selection of Shiga toxigenic Escherichia coli (STEC) isolates grown in broth and agar culture. Methods and Results:, Cellular surface charge was determined using zeta potential measurements. Hydrophobicity of the isolates was determined using bacterial adhesion to hydrocarbons assay, hydrophobic interaction chromatography and contact angle measurements. Microbial adhesion to solvents was used to determine the electron donor/acceptor characteristics. No differences of surface charge measurements were found between broth and agar grown cultures. Isolates belonging to serogroup O157 and serotypes O26:H11 and O111:H- were significantly (P < 0·05) less negatively charged than other STEC serotypes tested. All strains were hydrophilic with most methods and demonstrated a lower hydrophobicity in agar culture compared with broth culture. All strains demonstrated a strong microbial adhesion to chloroform indicating that STEC possess an electron donor and basic character. A relationship between serogroup O157 and other STEC serotypes was apparent using principal-component analysis (PCA). Conclusions:, Combining the results for physicochemical properties using PCA differentiated between strains belonging to the O157 serogroup and other STEC/non-STEC strains. PCA found similar results for broth and agar grown cultures. Significance and Impact of the Study:, Particular serotypes of STEC possess similar physicochemical properties which may play a role in their pathogenicity or potential attachment to various surfaces. [source] Protein purification using chromatography: selection of type, modelling and optimization of operating conditionsJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2009J. A. Asenjo Abstract To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, it is necessary to use several chromatographic steps. There is a range of techniques available including anion and cation exchange, which can be carried out at different pHs, hydrophobic interaction chromatography, gel filtration and affinity chromatography. In the case of a complex mixture of partially unknown proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98%, 99.5% or 99.9%). This review shows how an initial 'proteomic' characterization of the complex mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a specific level of purity with a minimum number of steps. The chosen methodology was implemented in a computer- based Expert System. Two algorithms were developed, the first algorithm was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants and the second algorithm was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The application of the Expert System approach was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with a supernatant of Bacillus subtilis producing a recombinant , -1,3-glucanase. Once the type of chromatography is chosen, optimization of the operating conditions is essential. Chromatographic elution curves for a three-protein mixture (, -lactoalbumin, ovalbumin and , -lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. In order to optimize operating conditions, a cost function was built that included the effect of the different production stages, namely fermentation, purification and concentration. This cost function was also successfully used for the determination of the fraction of product to be collected (peak cutting) in chromatography. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters. Copyright © 2008 John Wiley & Sons, Ltd. [source] New approaches for predicting protein retention time in hydrophobic interaction chromatography,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006M. E. Lienqueo Abstract Hydrophobic interaction chromatography (HIC) is an important technique for the purification of proteins. In this paper, we review three different approaches for predicting protein retention time in HIC, based either on a protein's structure or on its amino-acidic composition, and we have extended one of these approaches. The first approach correlates the protein retention time in HIC with the protein average surface hydrophobicity. This methodology is based on the protein three-dimensional structure data and considers the hydrophobic contribution of the exposed amino acid residues as a weighted average. The second approach, which we have extended, is based on the high correlation level between the average surface hydrophobicity of a protein's hydrophobic interacting zone and its retention time in HIC. Finally, a third approach carries out a prediction of the average surface hydrophobicity of a protein, using only its amino-acidic composition, without knowing its three-dimensional structure. These models would make it possible to test different operating conditions for the purification of a target protein by computer simulations, and thus make it easier to select the optimal conditions, contributing to the rational design and optimization of the process. Copyright © 2006 John Wiley & Sons, Ltd. [source] Hydrophilic interaction LC of peptides: Columns comparison and clusteringJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010Sylvia Van Dorpe Abstract A wide variety of hydrophilic interaction chromatography (HILIC) stationary phase surface chemistries are currently available. Although their selectivity can be considerably different, column comparison or clustering using peptides is limited. In this study, ten pharmaceutically relevant model peptides are analyzed on seven different HILIC columns (bare silica, amide, poly-hydroxyethyl aspartamide, diol and zwitterionic) for the evaluation of their performance and classification. The responses examined include single and multiple responses: plate number, asymmetry factor, LOD, geometric mean resolution, resolution product, time corrected resolution product, peak capacity and chromatographic response function. Column classification was performed using hierarchical clustering and principal component analysis. Moreover, the overall performance quality of the HILIC columns was compared using a linear desirability function. Hierarchical clustering and principal component analysis showed consistent clusters. The zwitterionic phase was clustered apart from the other HILIC columns and both poly-aspartamide columns were clustered together. In addition, the two bare silica phases represent two different clusters, and thus different selectivities. Overall, the responses showed the best performance for one of the bare silica columns (Alltima-Alltech), followed by the zwitterionic phase (ZIC)-HILIC. Thus, these columns, belonging to different clusters, were found to be the best performing systems in pharmaceutical peptide analysis for the selected peptide set. [source] Influence of stationary phase chemistry and mobile-phase composition on retention, selectivity, and MS response in hydrophilic interaction chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010Kenneth J. Fountain Abstract A comprehensive retention and selectivity characterization of several hydrophilic interaction chromatography (HILIC) stationary phases was performed with 28 test probes in order to study the influence of particle type, surface chemistry, and mobile-phase pH on chromatographic retention, selectivity, and MS response. Selectivity differences were compared for columns operated at both low and high pH, while ESI-MS was used to study the effects of mobile-phase pH on signal response. Additionally, acetone was explored as a potential alternative to ACN as the weak HILIC solvent. Moderate differences in selectivity were observed on the same column operated at different pH, mostly due to acidic compounds. In addition, the MS response increased when a high pH mobile phase was used, particularly for analytes that were ionized with negative ESI-MS. Even larger selectivity differences were observed for different stationary phases evaluated with the same mobile phase. Acetone was not a suitable replacement for ACN in routine HILIC separations due to differences in selectivity and MS response. Finally, the data from this study were used to establish guidelines for rapid HILIC method development of polar compounds, which is demonstrated with a mixture of histidine dipeptides and organophosphonate nerve agent metabolites. [source] Retention of arsenic species on zwitterionic stationary phase in hydrophilic interaction chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010Dan Xie Abstract Zwitterionic hydrophilic interaction chromatography (ZIC® -HILIC) was used to study the retention of selected organoarsenicals. The retention behavior of nine organic arsenic species on ZIC® -HILIC was investigated to elucidate which is the driving force for their separation, hydrophilic partitioning or adsorption driven by hydrogen bonds with surface H-donor/acceptor groups of the stationary phase. For this, the retention factor of the compounds k was correlated with log PO/W and with the calculated strength of hydrogen bonding of the analytes. By examining aliphatic and phenylic compounds separately, improved correlation was received. This indicates that both phenomena contribute to the separation of these arsenic species on ZIC® -HILIC. The results obtained evidence that considerable electrostatic interactions also occur on ZIC® -HILIC. Retention behavior of arsenic species was investigated by varying the separation conditions, which shows that the composition of the eluent has a strong influence on the retention behavior. It is highly dependent on water/acetonitrile ratio, pH value and salt additives. Dissociation degree and polarity of arsenic species, which are varying with pH, regulate the distribution of arsenic species between stationary and mobile phases in HILIC. Increase in the ammonium acetate concentration leads to shortened or to prolonged retention depending on the structure of the arsenic species. [source] Characterization of sialylated and fucosylated glycopeptides of ,2-glycoprotein I by a combination of HILIC LC and MALDI MS/MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010Akira Kondo Abstract Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N -glycans at each individual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter-ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human ,2-glycoprotein I (,2-GPI, apolipoprotein H), which contains multiple N -glycosylation sites. Conditions for rapid proteolytic digestion of ,2-GPI using low-specificity proteases were optimized to detect ,2-GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and tri-antennary complex type glycans, were characterized at three N -glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of ,2-GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein samples in functional proteomic research and advance our understanding of the biological role of protein glycosylation. [source] Monitoring of mutarotation of monosaccharides by hydrophilic interaction chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010ķ Pazourek Abstract Calibration based on the "single-point calibration method", a simple exponential transformation of the response function of an evaporative light scattering detector was improved and applied to analysis of selected saccharides under hydrophilic interaction chromatography mode (a polar phase LiChrospher100 DIOL, mobile phase acetonitrile/water). The improved approach to the calibration procedure yielded a calibration curve with an excellent linearity (quality coefficient <5%). This quantitative evaluation of chromatograms of D -galactose suggested that not only anomers but even pyranose and furanose forms of the anomers could be resolved , the resulting calculations of abundance of the anomeric form strongly correlated with data from the literature obtained mostly by NMR studies (analogous results were also obtained for D -arabinose, D -glucose, and D -mannose). Because of the rapid separation (retention time less than 10,min), the observed correlation enabled to monitor anomeric conversion (mutarotation) of monosaccharides. [source] Lactic acid quantitation in hand dishwashing liquid using an HILIC-UV methodologyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010Mark Storton Abstract Different hydrophilic interaction chromatography (HILIC) columns were screened for lactic acid separation in hand dishwashing liquid products and the influence of mobile phase strength, buffer concentration and column temperature on the retention of lactic acid on a Zorbax NH2 column was investigated. An isocratic HILIC method for the quantitation of lactic acid in hand dishwashing liquid products was developed. The mobile phase consists of 70% methanol and 30% 20,mM sodium phosphate buffer (v/v) at pH 2.5. The HILIC stationary phase is Zorbax NH2, 250×4.6 with a 5,,m particle size. Detection was carried out using a variable wavelength UV-VIS detector at 226,nm. The linear range and percent recovery for lactic acid in the products were 44.68,1206.39,,g/mL and 100.3%, respectively. This paper provides an optimized HILIC methodology for the analysis of an acidic polar analyte (lactic acid) on a basic stationary phase. The proposed method can be used for the routine analysis of lactic acid. [source] Rapid analysis of constituents of Radix Cyathulae using hydrophilic interaction-reverse phase LC-MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 22 2009Mei-Ting Ren Abstract A hydrophilic interaction chromatography (HILIC) and reverse-phase liquid chromatography (RPLC) coupled with electrospray TOF MS method was developed for the analysis and characterization of constituents in the radix of Cyathula officinalis Kuan. Separation parameters of HILIC such as buffer pH, mobile phase strength, and organic modifier were evaluated. Fructose, glucose, and sucrose were identified by HILIC-ESI/TOF MS. Reverse-phase liquid chromatography-ESI/TOF MS were applied for quick and sensitive identification of major saponins in Cyathula officinalis. In-source collision-induced dissociation has been performed to elucidate the fragmentation pathways of oleanane-, hederagenin-, and gypsogmin-type saponins. Twelve saponins were characterized in this plant for the first time, and four of them were presumed to be new compounds. In addition, one phytoecdysteroid (cyasterone) and one coumarin (6,7-dimethoxycoumarin) were detected at the same time. The present method was capable of rapid characterizing and providing structure information of constituents from herbal drugs. [source] Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 13 2009Damien L. Callahan Abstract Highly polar metabolites, such as sugars and most amino acids are not retained by conventional RP LC columns. Without sufficient retention low concentration compounds are not detected due ion suppression and structural isomers are not resolved. In contrast, hydrophilic interaction chromatography (HILIC) and aqueous normal phase chromatography (ANP) retain compounds based on their hydrophilicity and therefore provides a means of separating highly polar compounds. Here, an ANP method based on the diamond hydride stationary phase is presented for profiling biological small molecules by LC. A rapid separation system based upon a fast gradient that delivers reproducible chromatography is presented. Approximately 1000 compounds were reproducibly detected in human urine samples and clear differences between these samples were identified. This chromatography was also applied to xylem fluid from soyabean (Glycine max) plants to which 400 compounds were detected. This method greatly increases the metabolite coverage over RP-only metabolite profiling in biological samples. We show that both forms of chromatography are necessary for untargeted comprehensive metabolite profiling and that the diamond hydride stationary phase provides a good option for polar metabolite analysis. [source] Mixed-mode ion-exchangers and their comparative chromatographic characterization in reversed-phase and hydrophilic interaction chromatography elution modesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2008Michael Lämmerhofer Abstract A set of particulate silica-supported mixed-mode RP/weak anion-exchangers (RP/WAX) (obtained by bonding of N -undecenoylated 3-aminoquinuclidine, 3-aminotropane and 2-dimethylaminoethylamine as well as of N -butenoyl-(2S,4S,5R)-2-aminomethyl-5-[(2-octylthio)ethyl]-quinuclidine to thiol-modified silica) were chromatographically characterized in comparison to selected commercially available columns using two distinct isocratic elution modes, viz. an aqueous-rich RP-type elution mode (with 40% ACN and 60% buffer) as well as an organic solvent-rich hydrophilic interaction chromatography (HILIC)-type elution mode (95 and 90% ACN). The mixed-mode RP/WAX phases showed multimodal applicability, unlike a polar embedded RP material (Synergi Fusion RP), amino phases (Luna NH2, BioBasic AX) or typical HILIC packings (ZIC-HILIC, TSKGel Amide-80). Principal component analysis (PCA) of the RP test data confirmed that the in-house developed RP/WAX columns as well as the Acclaim Mixed-Mode WAX-1 phase resemble each other in their chromatographic characteristics having slightly lower hydrophobic selectivity (,CH2 of 1.5) than the tested Synergi Fusion RP (,CH2 ,1.8). In contrast, a decrease in mixed-mode character due to lowered ion-exchange capacity and concomitantly increased RP-like behavior could be identified for other mixed-mode phases in the order of Obelisc R > Primesep B2 > Uptisphere MM3. PCA on HILIC data revealed that the RP/WAX phases behave dissimilar to TSKGel Amide-80, ZIC-HILIC and polysulfoethyl A under the chosen elution conditions. Hence, they may be regarded as complementary to these commercial stationary phases with applicability profiles for hydrophilic but also hydrophobic solutes. [source] Evaluation of mobile phase, ion pairing, and temperature influence on an HILIC-MS/MS method for L -arginine and its dimethylated derivatives detectionJOURNAL OF SEPARATION SCIENCE, JSS, Issue 13 2008Giuseppe Paglia Abstract Asymmetric NG,,NG -dimethylarginine (ADMA) increases in diseases such as renal failure, diabetes mellitus, and hypercholesterolemia. The feasibility and utility of a hydrophilic interaction chromatography (HILIC) method for the separation of free L -arginine (Arg), ADMA, and symmetric NG,,NG, -dimethylarginine (SDMA) on a typical silica column were explored and the impact of some experimental parameters on the chromatographic behavior of these analytes was investigated. The effect of water and TFA content in mobile phase and of column temperature was investigated during the development of a fast and simple HILIC-MS/MS method that might be suitable for the quantification of free Arg, ADMA, and SDMA in plasma for routine analysis. Our results show that a good compromise between efficiency and peak shape with acceptable retention and total chromatographic run time is achieved using an ACN/water (90:10) mobile phase with TFA% as additive ranging from 0.015 to 0.025% and column temperature ranging from 25 to 30°C. [source] Preparation of a sorbitol methacrylate grafted silica as stationary phase for hydrophilic interaction chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2008Jonas Persson Abstract A new highly hydrophilic stationary phase based on graft polymerization of sorbitol methacrylate from the surface of Kromasil silica particles is described. Polymerization was initiated by thermal cleavage of tert -butyl hydroperoxide covalently attached to the silica particle surface. Due to the highly amphiphilic properties of the monomer, an extensive search was needed to find solvent conditions that enabled surface-initiated polymerization. This was finally solved by using a mixture of solvents that only partially dissolved the monomer. The graft polymerization was confirmed by Fourier transform infrared spectroscopy and elemental analysis. The resulting stationary phase was evaluated by HPLC and exhibited a selectivity markedly different from that of commercially available columns and of neat silica. [source] The application of novel 1.7 ,m ethylene bridged hybrid particles for hydrophilic interaction chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2008Eric S. Grumbach Abstract An un-derivatized 1.7 ,m ethylene bridged hybrid (BEH) particle was evaluated for its utility in retaining polar species in hydrophilic interaction chromatography (HILIC), and was compared to a 3 ,m un-derivatized silica material. Retentivity as a function of mobile phase pH, polar modifier and ACN content was examined. Also, the efficiency of the two particle substrates was compared by plotting HETP vs. linear velocity. Improved chemical resistance of the un-derivatized BEH particle was compared to un-derivatized silica at pH 5, demonstrating no performance deterioration over the course of 2000 injections for the BEH particle, while the silica particle deteriorated rapidly after 800 injections. Lastly, ESI-MS sensitivity as a function of particle size and separation mode was demonstrated. A 2.2 to 4.7-times higher ESI-MS response was observed on the 1.7 ,m particle compared to the 3 ,m particle, whereas a 5.6 to 8.8-times higher ESI-MS response was observed using HILIC as when compared to traditional RP chromatography. [source] Hydrophilic interaction and reversed-phase ultra-performance liquid chromatography TOF-MS for metabonomic analysis of Zucker rat urineJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2008Helen G. Gika Abstract Hydrophilic interaction chromatography (HILIC) provides a complementary technique to RP methods for the retention of polar analytes for LC-MS-based metabonomic studies. Combining the advantages of both RP and HILIC separations with the efficient and rapid separations obtained using sub-2 ,m particles via the recently introduced ultra-performance LC (UPLC) enables increased coverage of the metabolites present in biological samples to be achieved. Here an HILIC-UPLC-MS method was developed to provide metabolite profiles for urine samples obtained from male Zucker rats. The resulting data were compared with results obtained for the same samples by RP-UPLC-MS and demonstrated the complementary nature of the two separations with both methods enabling discrimination between the different sample types. Interestingly sample type differentiation was based on different markers. [source] Stability-indicating assay of sodium cromoglicate in ophthalmic solution using mixed-mode hydrophilic interaction chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2008Mohammed Shahid Ali Abstract A hydrophilic interaction chromatographic (HILIC) procedure for the quantification of Sodium Cromoglicate (SCG) in ophthalmic solution is developed. Mobile phase consists of ACN and buffer, 86:14 v/v. Atlantis HILIC,Si column, 25 cm×4.6 mm, is used as stationary phase. Detection is carried out using a variable wavelength UV-Vis detector at 326 nm. Linearity range and percent recoveries for SCG were 50,400 ,g/mL and 100.44%, respectively. The SCG HILIC-UV assay was validated according to the International Conference on Harmonization guidelines. The method separates two impurities and degradation products resulting from stress environment. Influence of organic solvent, ionic strength and mobile phase pH on the retention of SCG is studied. The paper provides optimization of polar anionic solute (SCG) on unmodified silica by HILIC. Proposed method can be used as a stability-indicating assay for SGC and can be proved to be beneficial in ESI-MS for enhanced sensitivity. [source] Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2004Augustine C Ogbonna Abstract A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion-exchange chromatography on Q- and S-Sepharose (fast flow), gel filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The enzyme was purified 8.4-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg,1 protein. SDS,PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0,8.0 and retained over 60% activity at 70 °C after 30-min incubation. It was highly significantly (P < 0.001) inhibited by Hg2+, appreciably (P < 0.01) inhibited by Ag+, Ba2+ and Pb2+ but highly significantly (P < 0.001) activated by Co2+, Mn2+ and Sr2+. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p -chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: Km = 0.33 mg ml,1; Vmax = 0.08 µmol ml,1 min,1. Copyright © 2004 Society of Chemical Industry [source] Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celeryMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 12 2007Mirko A. Bollen Abstract Birch pollen allergy is predominantly caused by the major allergen Bet v 1 and can lead to crossreactions with homologous proteins in food. Two major cross-reactive food allergens are Dau c 1 from carrot and Api g 1 from celery, which have never been purified from their natural source. Here, we describe a non-denaturing purification method for obtaining natural Bet v 1, Dau c 1 and Api g 1, comprising of ammonium sulfate precipitation, hydrophobic interaction chromatography and size exclusion chromatography. This method resulted in 98,99% pure isoform mixtures for each allergen. Characterization of these isoform mixtures with Q-TOF MS/MS clearly showed earlier reported isoforms of Bet v 1, Dau c 1 and Api g 1, but also new isoforms. The presence of secondary structure in the three purified allergens was demonstrated via circular dichroism and showed high similarity. The immune reactivity of the natural allergens was compared with recombinant proteins by Western blot and ELISA and showed similar reactivity. [source] Purification and partial characterization of a dipeptidyl peptidase from Prevotella intermediaMOLECULAR ORAL MICROBIOLOGY, Issue 3 2003Y. Shibata A peptidase hydrolyzed X-Pro- p -nitroanilide was purified from the cell extract of Prevotella intermedia ATCC 25611 by ion-exchange chromatography and hydrophobic interaction chromatography. The purified enzyme exhibited a molecular size of 74 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the maximum enzyme activity was found between pH 7.0 and pH 7.5. This peptidase was a serine enzyme and hydrolyzed Lys-Pro- p -nitroanilide, Arg-Pro- p -nitroanilide, and Ala-Pro- p -nitroanilide, but Lys-Ala- p -nitroanilide was not split. The enzyme may be classified as a dipeptidyl peptidase IV. [source] Purification and characterisation of two ACC oxidases expressed differentially during leaf ontogeny in white cloverPHYSIOLOGIA PLANTARUM, Issue 1 2000Deming Gong Two isoforms of ACC oxidase (ACO) (EC 1.4.3), expressed differentially during leaf ontogeny in white clover (Trifolium repens L.), have been identified and purified to homogeneity. One isoform, designated MGI, was purified from mature green leaf tissue while the second isoform, designated SEII, was purified from senescent leaf tissue. The isolation and purification of these isoforms were achieved using a combination of hydrophobic interaction chromatography, anion exchange chromatography, chromatofocusing and gel filtration column chromatography. The Mr of both MGI and SEII was determined to be 37.5 kDa by gel filtration, and 37 kDa (MGI), 35 kDa (SEII) by SDS-PAGE, indicating that both isoforms are active as monomers. During purification, both isoforms were recognised by a polyclonal antibody directed against a recombinant polypeptide derived from a white clover ACO gene expressed in mature green leaf tissue, TR-ACO2. In addition to molecular mass, differences between the two isoforms were observed in terms of pH optima, isoelectric point (pI), Km for ACC, optimal requirements for the co-substrate ascorbate, and NaHCO3 and Fe2+ as co-factors. The identification of distinct ACC oxidases from the same tissue at different developmental stages shows that the now widely observed transcriptional regulation of the ACO gene family in higher plants is also expressed in terms of differential regulation of enzyme isoforms. [source] Probing genetic algorithms for feature selection in comprehensive metabolic profiling approachRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 8 2008Wei Zou Six different clones of 1-year-old loblolly pine (Pinus taeda L.) seedlings grown under standardized conditions in a green house were used for sample preparation and further analysis. Three independent and complementary analytical techniques for metabolic profiling were applied in the present study: hydrophilic interaction chromatography (HILIC-LC/ESI-MS), reversed-phase liquid chromatography (RP-LC/ESI-MS), and gas chromatography all coupled to mass spectrometry (GC/TOF-MS). Unsupervised methods, such as principle component analysis (PCA) and clustering, and supervised methods, such as classification, were used for data mining. Genetic algorithms (GA), a multivariate approach, was probed for selection of the smallest subsets of potentially discriminative classifiers. From more than 2000 peaks found in total, small subsets were selected by GA as highly potential classifiers allowing discrimination among six investigated genotypes. Annotated GC/TOF-MS data allowed the generation of a small subset of identified metabolites. LC/ESI-MS data and small subsets require further annotation. The present study demonstrated that combination of comprehensive metabolic profiling and advanced data mining techniques provides a powerful metabolomic approach for biomarker discovery among small molecules. Utilizing GA for feature selection allowed the generation of small subsets of potent classifiers. Copyright © 2008 John Wiley & Sons, Ltd. [source] On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2005Jörgen Bengtsson A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5,mM ammonium acetate (70:30) for separation. Microdialysis samples (5,µL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55,ng/mL, respectively, and the method was linear from LLOQ to 200,ng/mL. For plasma, a volume of 100,µL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1,ng/mL and the ranges were 2.0,2000 and 3.1,3100,ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53,ng/mL and the ranges were 0.78,500, 1.49,1000 and 0.53,500,ng/mL for morphine, M3G and M6G, respectively. Copyright © 2005 John Wiley & Sons, Ltd. [source] Simple and rapid determination of 1-deoxynojirimycin in mulberry leavesBIOFACTORS, Issue 1-4 2004Toshiyuki Kimura Abstract A simple and rapid method for determining 1-deoxynojirimycin (DNJ), a potent glucosidase inihibitor present in mulberry leaves (Morus alba and Morus bombysis), by high performance liquid chromatography coupled to an evaporative light scattering detector (ELSD) has been developed. DNJ was separated from extract of mulberry leaves on TSK gel Amide-80 column, which is a representative column for hydrophilic interaction chromatography. During post column detection, DNJ was detected by ELSD and concurrently identified by mass spectrometry. The detection limit was 100 ng. This method is sufficiently sensitive for determining DNJ in mulberry leaves and other related products. [source] Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regressionBIOPOLYMERS, Issue 11 2008Yeqiu Wang Abstract The secondary structures of proteins (,-helical, ,-sheet, ,-turn, and random coil) in the solid state and when bound to polymer beads, containing immobilized phenyl and butyl ligands such as those as commonly employed in hydrophobic interaction chromatography, have been investigated using FTIR-ATR spectroscopy and partial least squares (PLS) methods. Proteins with known structural features were used as models, including 12 proteins in the solid state and 7 proteins adsorbed onto the hydrophobic surfaces. A strong PLS correlation was achieved between predictions derived from the experimental data for 4 proteins adsorbed onto the phenyl-modified beads and reference data obtained from the X-ray crystallographic structures with r2 values of 0.9974, 0.9864, 0.9924, and 0.9743 for ,-helical, ,-sheet, ,-turn, and random coiled structures, respectively. On the other hand, proteins adsorbed onto the butyl sorbent underwent greater secondary structural changes compared to the phenyl sorbent as evidenced from the poorer PLS r2 values (r2 are 0.9658, 0.9106, 0.9571, and 0.9340). The results thus indicate that the secondary structures for these proteins were more affected by the butyl sorbent, whereas the secondary structure remains relatively unchanged for the proteins adsorbed onto the phenyl sorbent. This study has important ramifications for understanding the nature of protein secondary structural changes following adsorption onto hydrophobic sorbent surfaces. This knowledge could also enable the development of useful protocols for enhancing the chromatographic purification of proteins in their native bioactive states. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 895,905, 2008. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] | ||||||||||||||||