Column characterization
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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.
Cíle studie: Cílem studie bylo vytvořit protokol pro zpracování vzorků moči pro získání aminokyselinových profilů pomocí iontově výměnné chromotografie s detekcí ve viditelném spektru (IEC/Vis) na základě tvorby komplexů aminokyselin s ninhydrinem a následné zkrácení času analýzy pro klinicky zajímavé molekuly (sarkosin či taurin), které mohou byt využity jako potenciální nádorové biomarkery. Typ studie: Metodická Materiál a metody: Vzorky moči, odebrané pacientům s diagnostikovaným karcinomem prostaty (n = 500) byly shromažďovány po dobu 1 roku a uskladňovány při teplotě -80°C. Pro vlastní analýzu aminokyselinových profilů byly vzorky připraveny kyselou hydrolýzou v mikrovlnném reaktoru. Za optimalizovaných podmínek (80 W, 120°C, 25 bar, 105 min) bylo 500 μl vzorku, smíchaného s 500 μl 35% kyseliny chlorovodíkové zhydrolyzováno na výsledný produkt, který byl následně ředěn pufrem sodíkového cyklu (0,2 mol/l NaCl, 60 mmol/l C6H807, 1,5 mmol/l N3Na a 0,4 % S(CH2CH2OH)2). Po centrifugaci (25 000 g při 4°C, 20 min) byly vzorky neutralizovány (0,6 mol/l NaOH) a analyzovány iontově-výměnnou kapalinovou chromatografií s postkolonovou derivatizací ninhydrinem využívající optickou detekci při vlnových délkách λ = 440 a 570 nm. Pro detekci sarkosinu a taurinu byly vzorky připraveny odpařením 250 μl vzorku na dusíkové odparce (40 min, teplota dusíku 60°C, tlak 1 bar) a následně resuspendovány pufrem sodíkového cyklu (250 μl). Výsledky a závěr: Analýzou aminokyselinových profilů (n = 500) bylo zjištěno, že lze docílit získání důležitých informací z neinvazivně odebraných vzorků moči. Použití dusíkové odparky redukuje manipulaci se vzorkem a zlepšuje opakovatelnost analýzy. Zkrácená analýza sarkosinu a taurinu ze vzorků odpařených na dusíkové odparce může sloužit jako metoda pro citlivou a nízkonákladovou analýzu klinických vzorků.
Objective: The aim of our study was to prepare a protocol for pre-treatment of clinical urinary samples, subsequently employed for acquirement of amino acid profiles via ion exchange chromatography with detection in visible spectrum (IEC/ Vis). Further for clinically interesting biomolecules as sarcosine and taurine the optimization was carried out to shorten the analysis time. Design: Methodological Material and methods: Urine specimens from patients diagnosed with prostate cancer (n = 500) were collected within 1 year and stored in -80°C. Samples were processed for analysis of amino acid profiles through acidic hydrolysis in a microwave reactor. Using optimized conditions (80 W, 120°C, 25 bar, 105 min), 500 μL sample, mixed with 500 μL of 35% hydrochloric acid hydrolyzed. That resulted in product, which was subsequently diluted in buffer of sodium cycle (0.2 mol/L NaCl, 60 C6H807 mmol/L, 1.5 mmol/L and 0.4% N3Na S(CH2CH2OH)2). After centrifugation (25000 g at 4°C, 20 min), the samples were neutralized (0.6 mol/l NaOH) and analyzed using ion-exchange liquid chromatography with post-column derivatization with ninhydrin, using the detection wavelength λ = 440 and 570 nm (IEC/Vis). For analysis of sarcosine and taurine, the samples were prepared by evaporation of 250 μL of sample employing a nitrogen evaporator (40 min, temperature 60°C. Nitrogen pressure of 1 bar) and resuspended with buffer of sodium cycle (250 μL). Results and conclusion: The analyses of amino acid profiles offer interesting clinical information not only in non-invasively collected urinary samples, but also in other organic matrices. Employment of nitrogen evaporator for sample pre-treatment leads to reduction of manipulation with sample and its combination with shortened analyses times of sarcosine and taurine may serve as a sensitive and low-cost method for analysis of clinical specimens.
- MeSH
- časná diagnóza * MeSH
- chromatografie iontoměničová * metody přístrojové vybavení využití MeSH
- lidé MeSH
- nádorové biomarkery analýza MeSH
- nádory prostaty * diagnóza MeSH
- oxidační stres MeSH
- plošný screening ekonomika využití MeSH
- prostatický specifický antigen * analýza MeSH
- sarkosin * analýza moč MeSH
- spektrofotometrie ultrafialová metody přístrojové vybavení využití MeSH
- taurin * analýza moč MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Derivatized cyclofructans have been recently introduced as a new class of chiral selectors with great application potential. In this study, a R-naphthylethyl-functionalized cyclofructan 6 based chiral stationary phase (RN CF6 CSP) was used for separation of substituted binaphthyl catalysts in the normal phase HPLC mode. Dominant interaction types that play a role in the separation mechanism were revealed by a linear free energy relationship (LFER) method. In order to evaluate the contribution of the substituent on the cyclofructan structure to retention, the R-naphthylethyl-functionalized β-cyclodextrin (RN CD) CSP was chosen for comparison. Retention factors of 46 widely different solutes, with known solvation parameters, were determined on each of the columns under the same mobile phase compositions used for the enantiomeric separations. The LFER results showed that hydrogen bond acidity and polarity/polarizibility have the greatest impact on retention and enantioresolution on the RN CF6 CSP. The equal influence of the naphthylethyl substituent on the both CSPs was also confirmed while the effects of the basic cyclofructan versus cyclodextrin structures were different. The addition of trifluoroacetic acid to the hexane/propane-2-ol mobile phase was negligible on the RN CF6 CSP for the majority of atropoisomers except for one with ionizable functional groups. The RN CF6 column was shown to be more suitable for enantioseparation of the binaphthyl catalysts than the RN CD column. Higher retention offered by the latter CSP had no positive effect on the enantioresolution.
OBJECTIVES: Candida tropicalis yeast is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This yeast is capable of utilizing phenol as the sole carbon and energy source. While the enzyme participating on the first step of phenol biodegradation, NADPH-dependent phenol hydroxylase, has already been characterized, information on the enzyme participating in the second step of its degradation, catechol 1,2-dioxygenase, is scarce. The development of the procedure suitable for catechol 1,2-dioxygenase isolation and partial characterization of this enzyme are the aims of this study. METHODS: Combination of chromatography on DEAE-Sepharose and gel-permeation chromatography on Sephadex G-100 was used for isolation of cytosolic catechol 1,2-dioxygenase from C. tropicalis yeast. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephadex G-100 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (catechol consumption and/or cis,cis-muconic acid formation). RESULTS: Using the isolation procedure consisting of chromatography and re-chromatography on a column of DEAE-Sepharose and gel filtration on Sephadex G-100, catechol 1,2-dioxygenase was purified from C. tropicalis cytosol to homogeneity. Catechol 1,2-dioxygenase was found to be a homodimer with a subunit molecular mass of 30000 +/- 5000. The enzyme oxidized catechol producing cis,cis-muconic acid. The optimal temperature and pH were 30 degrees C and 7.7, respectively. CONCLUSIONS: The data are the first report showing the isolation of eukaryotic catechol 1,2-dioxygenase from C. tropicalis to homogeneity and its partial characterization.
- MeSH
- Candida tropicalis enzymologie metabolismus MeSH
- chromatografie MeSH
- cytosol chemie enzymologie metabolismus MeSH
- dextrany MeSH
- dimerizace MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fenol chemie MeSH
- fungální proteiny chemie izolace a purifikace metabolismus MeSH
- gelová chromatografie MeSH
- katechol-1,2-dioxygenasa chemie izolace a purifikace metabolismus MeSH
- katecholy chemie metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- kyselina sorbová analogy a deriváty chemie metabolismus MeSH
- oxidace-redukce MeSH
- teplota MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX.
Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.
- MeSH
- diglyceridy krev izolace a purifikace metabolismus MeSH
- dospělí MeSH
- exozómy chemie metabolismus MeSH
- fosfatidylcholiny krev izolace a purifikace metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipidomika metody MeSH
- lysofosfatidylcholiny krev izolace a purifikace metabolismus MeSH
- metabolismus lipidů * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- superkritická fluidní chromatografie metody MeSH
- triglyceridy krev izolace a purifikace metabolismus MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn2+ and Mg2+ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg2+. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.
The foul odour of cheese-production wastewater is a common problem in areas surrounding dairy wastewater treatment plants. For successful odour management, a better understanding of the key odorants and how to handle them during wastewater treatment is needed. This paper documents the results of using gas chromatography-mass spectrometry coupled with olfactometry (GC-MS/O) to analyze odours emanating from a possibly overloaded treatment plant in Czechia. Using a DB5 capillary column, 20 compounds were detected and identified, nonanal (FDgeomean 152) and octen-3-ol (FDgeomean 2048) having the most pungent odours.
