The morphology, composition, and selectivity of a silica-based monolithic stationary phase, grafted by a layer of trioctyl(3/4-vinylbenzyl)phosphonium chloride ([P888VBn]Cl), is presented. The results of elemental analysis confirmed that the prepared stationary phase contains 38.8 at.% of silicon, 60.2 at.% of carbon, and 1.0 at.% of phosphorus. Capillary columns (150 × 0.1 mm) for liquid chromatography were evaluated using alkylbenzenes, monosubstituted benzenes, polyaromatic compounds, substituted benzene regioisomers, and aromatic carboxylic and amino acids. The prepared ionic liquid (IL)-based stationary phase exhibits hydrophobic, hydrophilic, and electrostatic interactions, as confirmed by experiments on the evaluation of the effect of the mobile phase composition (content of acetonitrile and ammonium formate) on the isocratic chromatographic separation. Thus, the IL-based capillary column demonstrates a unique separation selectivity compared to Phenyl-, C8-, and C18-stationary phases, and high efficiency for an expanding number of structurally diverse compounds.
New strategies for the fast, efficient, and environmentally friendly extraction of proteins are required to isolate desired bioactive compounds from a technological point of view. In this study, utilization of the pressurized water extraction (PWE) at low temperature (40 °C) for isolation of mistletoe proteins was investigated. PWE effectiveness, based on protein fingerprints, were compared with those obtained by conventional extractions using 10 mmol L-1 Tris-HCl buffer pH 8.3, 50 mmol L-1 phosphate buffer pH 7, or deionized water. The extracts were precipitated using acetone, trichloroacetic acid (TCA), and 20% (w/v) TCA/acetone and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PWE was more or equally efficient for isolation of mistletoe proteins than evaluated conventional extraction methods. The proteomic analysis combining mass spectrometry and database searching confirmed the presence of 35 proteins in PWE extracts precipitated by acetone, which was the most compounds identified from all studied extracts. The PWE high extraction power was revealed for multiple viscotoxin isoforms and specific enzymes indispensable for the synthesis of terpenes.
- MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- listy rostlin MeSH
- proteomika MeSH
- Viscum album * MeSH
- voda MeSH
- Publikační typ
- časopisecké články MeSH
The advantages of using mixtures of organic solvents for the separation of labeled oligosaccharides on the amide stationary phase under hydrophilic interaction liquid chromatography conditions are presented. The effect of the type of buffer as well as solvent or their mixtures on retention of uracil, saccharide labeling reagents (2-aminobenzoic acid, 2-aminobenzamide, ethyl 4-aminobenzoate, procainamide), and corresponding labeled saccharides were evaluated. The successful isocratic separation of labeled isomeric trisaccharides (maltotriose, panose, and isomaltotriose) was achieved in the mobile phase consisting of a 90% (v/v) mixture of organic solvents (methanol/acetonitrile 60:40) and 10% (v/v) 30 mM ammonium formate, pH 3.3. Changing the volume ratio between methanol/acetonitrile from 60:40 to 50:50 (v/v) allowed to obtain the separation of di-, tri-, and tetrasaccharides labeled by ethyl 4-aminobenzoate in less than 10.5 min.
- MeSH
- acetonitrily chemie MeSH
- amidy chemie MeSH
- chemické techniky analytické přístrojové vybavení metody MeSH
- chromatografie kapalinová * MeSH
- formiáty chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- isomerie MeSH
- oligosacharidy izolace a purifikace MeSH
- ortoaminobenzoáty chemie MeSH
- rozpouštědla chemie MeSH
- sacharidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
A novel and original application of salting-out assisted liquid-liquid extraction is presented. This technique was used to purify the final reaction products (quaternary ammonium salts) from unreacted components and by-products present in multiple excesses. The partition of two structurally related compounds as (2-aminoethyl)trimethylammonium salt (a labeling reagent) and a derivative of [2-(imidazoline-1-yl)ethyl]trimethylammonium salt (a final reaction product of N-acetylglucosamine labeling by (2-aminoethyl)trimethylammonium salt) between acetonitrile-rich and water-rich layers was monitored by hydrophilic interaction chromatography with electrospray ionization mass spectrometry. Despite the poor solubility of both highly polar substances in solutions containing a high concentration of acetonitrile, the main portion of the labeling reagent (72%) can be removed from the crude reaction mixture in the first extraction step using 95% acetonitrile/5% water as an extraction solvent. The purified final reaction product contained only 2% of the labeling reagent, and it was suitable for analysis by direct infusion mass spectrometry to confirm its identity. The capability of the suggested purification protocol to process small-volume highly salted reaction mixtures was also proven by analysis of saccharide mixture containing glucose, maltose, and maltotriose labeled by the positively charged tag.
- Publikační typ
- časopisecké články MeSH
A methodology for preparing phosphonium-based ionic liquid modified silica-based monolithic capillary columns is presented. The silica monolithic columns with dimensions of 150 × 0.1 mm were modified by a phosphonium-based ionic liquid (trioctyl(3/4-vinylbenzyl)phosphonium chloride) via 3-(trimethoxysilyl)propyl methacrylate. The prepared columns were evaluated under hydrophilic interaction liquid chromatography separation conditions, employing a sample mixture containing purine and pyrimidine bases and nucleosides. Detection was made by UV. The high efficiency of the original silica monolith was preserved even after modification, and it reached values in the range of 98,000-174,000 theoretical plates/m. The effects of the concentration of acetonitrile in the mobile phase, the presence of additives in the mobile phase, such as, acetic acid or ammonium acetate, and the pH of the mobile phase on the separation of some selected analytes were investigated. The prepared columns showed different separation selectivity compared to silica, phenyl and sulfobetaine stationary phases.
- MeSH
- betain analogy a deriváty chemie MeSH
- chromatografie kapalinová metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- iontové kapaliny chemická syntéza chemie MeSH
- nukleosidy analýza MeSH
- organofosforové sloučeniny chemie MeSH
- oxid křemičitý chemie MeSH
- poréznost MeSH
- reprodukovatelnost výsledků MeSH
- Publikační typ
- časopisecké články MeSH
The synthesis and characterization of large-bore silica-based monolithic capillary columns (0.32mm×150mm) are presented. Columns were prepared by acidic hydrolysis of a mixture containing tetramethoxysilane (TMOS) and 1,2-bis(trimethoxysilyl)ethane (BTME) in different molar ratios in the presence of polyethylene glycol and urea. The monoliths were modified by zwitterionic monomer [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide via 3-(trimethoxysilyl)propyl methacrylate. Prepared stationary phases were evaluated by scanning electron microscopy and chromatographic separation of nucleobases and their derivatives in the HILIC mode. The best chromatographic results were obtained with the column prepared from the reaction mixture containing BTME and TMOS in a 1:4 molar ratio. The permeability of such column reached 1.68×10(-14)m(2) and the efficiency, expressed as a height equivalent of the theoretical plate, did not exceed 10.5μm for the tested compounds. The columns were successfully applied to HILIC separation of native and labeled oligosaccharides and glycans released from bovine ribonuclease B and human immunoglobulin G.
- MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- ethan analogy a deriváty chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hydrofobní a hydrofilní interakce MeSH
- imunoglobulin G metabolismus MeSH
- lidé MeSH
- methakryláty chemie MeSH
- mikroskopie elektronová rastrovací MeSH
- oligosacharidy analýza izolace a purifikace MeSH
- organické sloučeniny křemíku chemie MeSH
- oxid křemičitý chemie MeSH
- ribonukleasy metabolismus MeSH
- skot MeSH
- trimethylsilylové sloučeniny chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The methicillin-resistant Staphylococcus aureus causes difficult-to-treat healthcare-associated infections in humans. For fast and effective selection of an appropriate antibiotic therapy, it is essential to have rapid and reliable methods for differentiation of methicillin-resistant S. aureus from less dangerous methicillin-sensitive S. aureus. There have been many methods for the identification of methicillin-resistant S. aureus described but none has been accepted as an international standard. The most commonly used techniques such as phenotyping and genotyping have a few disadvantages, for instance, these techniques are not reproducible and stable. In addition, they are time-consuming, expensive, and they are not capable to distinguish all S. aureus strains. In this study, the methicillin-resistant and methicillin-sensitive S. aureus isolates obtained from patients were extracted in hot water. The released proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing. These two methods were able to differentiate among tested bacterial strains. The proposed methods are time saving, they are applicable in standard biochemical laboratories, and they do not require any expensive equipment.
- MeSH
- bakteriologické techniky metody MeSH
- časové faktory MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- isoelektrická fokusace metody MeSH
- lidé MeSH
- rezistence na methicilin * MeSH
- Staphylococcus aureus chemie klasifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Here, we have reviewed separation studies utilizing monolithic capillary columns for separation of compounds preceding MS analysis. The review is divided in two parts according to the used separation method, namely CEC and capillary LC (cLC). Based on our overview, monolithic CEC-MS technique have been more focused on the syntheses of highly specialized and selective separation phase materials for fast and efficient separation of specific types of analytes. In contrast, monolithic cLC-MS is more widely used and is often employed, for instance, in the analysis of oligonucleotides, metabolites, and peptides and proteins in proteomic studies. While poly(styrene-divinylbenzene)-based and silica-based monolithic capillaries found their place in proteomic analyses, the other laboratory-synthesized monoliths still wait for their wider utilization in routine analyses. The development of new monolithic materials will most likely continue due to the demand of more efficient and rapid separation of increasingly complex samples.
- MeSH
- hmotnostní spektrometrie * MeSH
- kapilární elektrochromatografie * MeSH
- lidé MeSH
- myši MeSH
- oligonukleotidy analýza MeSH
- proteiny analýza MeSH
- žlučové kyseliny a soli analýza MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The history of liquid chromatography started more than a century ago and miniaturization and automation are two leading trends in this field. Nanocolumn liquid chromatography (nano LC) and largely synonymous capillary liquid chromatography (capillary LC) are the most recent results of this process where miniaturization of column dimensions and sorbent particle size play crucial role. Very interesting results achieved in the research of extremely miniaturized LC columns at the end of the last century lacked distinctive raison d'être and only advances in mass spectrometry brought a real breakthrough. Configuration of nano LC-electrospray ionization mass spectrometry (LC-ESI-MS) has become a basic tool in bioanalytical chemistry, especially in proteomics. This review discusses and summarizes past and current trends in the realization of nano liquid chromatography (nano LC) platforms. Special attention is given to the mobile phase delivery under nanoflow rates (isocratic, gradient) and sample injection to the nanocolumn. Available detection techniques applied in nano LC separations are also briefly discussed. We followed up the key themes from the original scientific reports over gradual improvements up to the contemporary commercial solutions.
In this study a strategy to immobilize phospholipids onto a polymer-based stationary phase is described. Methacrylate-based monoliths in capillary format (150×0.1mm) were modified by soybean phosphatidylcholine through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling to obtain stationary phases suitable to mimic cell surface membranes. The covalent coupling reaction involves the phosphate group in phospholipids; therefore, the described methodology is suitable for all types of phospholipids. Immobilization of soy bean phosphatidylcholine on the monolith was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry of the fatty alcohol profile, generated upon reductive cleavage of the fatty acyl side chains of the phospholipid on the monolith surface with lithium aluminium hydride. The prepared stationary phases were evaluated through studies on the retention of low-molar mass model analytes including neutral, acidic, and basic compounds. Liquid chromatographic studies confirmed predominant hydrophobic interactions between the analytes and the synthesized stationary phase; however, electrostatic interactions contributed to the retention as well. The synthesized columns showed high stability even with fully aqueous mobile phases such as Dulbecco's phosphate-buffered saline solution.
- MeSH
- biomimetika MeSH
- chemické techniky analytické přístrojové vybavení metody MeSH
- chromatografie kapalinová přístrojové vybavení MeSH
- ethyldimethylaminopropylkarbodiimid chemie MeSH
- fosfatidylcholiny chemie MeSH
- fosfolipidy chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- methakryláty chemie MeSH
- polymery chemie MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
