A new, rapid, sensitive, robust, and reliable method has been developed for the qualitative analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate using gas chromatography with mass spectrometry and two-step trimethylsilylation. The method employs hexamethyldisilazane for silylation of the phosphate and hydroxyl groups in the first phase and bis(trimethylsilyl)trifluoroacetamide for silylation of the less-reactive amino groups in the second phase. This order is of key importance for the method because of the different reactivities of the two reagents and the mechanism of derivatization of the active groups of the analytes. Trimethylsilylated derivatives of the analytes were identified on the basis of their retention times and mass spectra. The probable structures of the major fragments were identified in the spectra of the trimethylsilylated derivatives and characteristic m/z fragments were selected for each analyte. Fragments with m/z 73 and 299 occurred in the spectra of all the analytes. The characteristic retention data were employed to calculate the retention indices of the individual silylated phosphorylated substances in the hydrocarbon range C12-C19 for the DB-5ms column. The method was employed to measure the polar fraction of the hydrolysate of the cytoplasmic membrane of Bacillus subtilis. The detection limits vary between 5 μg/mL (trimethylsilylated phosphate) and 72 μg/mL (trimethylsilylated phosphoethanolamine).
The methodology for separations of saccharides in standard electrophoretic systems has been transferred to the short-capillary electrophoresis format. The laboratory-designed apparatus used employs a quartz capillary with an internal diameter of 10 μm, a total length of 10 cm, and an effective length of 4 cm, in combination with contactless conductivity detection. It has been applied to separations of neutral mono- and disaccharides. The saccharides are separated in the anionic form, in solutions of alkali hydroxides, namely, KOH, NaOH, and LiOH. The separation of a model mixture of five saccharides (sucrose, lactose, glucose, fructose, and ribose) takes less than 1 min, the LOD equaling 15, 35, 19, 17, and 24 mg L(-1) and the LOQ equaling 52, 117, 63, 53, and 79 mg L(-1) for sucrose, lactose, glucose, fructose, and ribose, respectively. The technique developed has been used to determine sucrose, glucose and fructose in high-energy drinks. The separation is finished within less than 50 s; the saccharide contents determined are identical with the declared values within the reliability interval in most cases, the RSD value being mostly less than 2%. In general, the separation system developed is very convenient for rapid analyses of large sets of similar samples, e.g., in product quality control or environmental monitoring.
- MeSH
- časové faktory MeSH
- design vybavení MeSH
- elektrická vodivost MeSH
- elektroforéza kapilární ekonomika přístrojové vybavení MeSH
- energetické nápoje analýza MeSH
- limita detekce MeSH
- monosacharidy analýza izolace a purifikace MeSH
- sacharosa analýza izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.
- MeSH
- chromatografie micelární elektrokinetická kapilární metody MeSH
- elektroforéza kapilární metody MeSH
- enzymatické testy metody MeSH
- isoelektrická fokusace metody MeSH
- izotachoforéza metody MeSH
- kapilární elektrochromatografie metody MeSH
- kinetika MeSH
- lidé MeSH
- miniaturizace metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
In proteomics, proteins can be identified by enzymatic cleavage of the target protein using an enzyme of the known specificity (primarily trypsin), sequencing the obtained specific peptides by MS and comparing the amino acid sequence of the peptides with a protein database. The sophisticated approach described above was used in this study to determine and verify the original species of cheeses. Proteins were extracted from three different cheese samples which were produced from cow, sheep and goat milks. The isolated proteins were cleaved with trypsin and the peptides obtained were sequenced and identified by a HPLC-chip/MS/MS microfluidic system. Two different extraction methods and two various chromatographic sorbents packed in plastic chips were studied. Beta-lactoglobulin and four kinds of casein were found in the cheese samples. The species of kappa-casein were identified unambiguously in all the three cheese samples and, thus, kappa-casein can be used to determine the origin of milk of the cheese. The other proteins found in the samples show very similar primary structures and cannot be recommended for identification of the cheese milk origin.
Polymeric macromolecules of well-designed structures and specific properties open promising directions in the capillary entangled polymer electrophoresis. Pluronic F-127, as a thermoassociating polymer, possesses some unique properties that can be utilized in capillary entangled polymer electrophoresis of amino acids, peptides and proteins. In this study, we studied properties of Pluronic F-127 polymer as an additive to BGE for the separation of peptides and proteins. The influence of the thermoassociation on separation selectivity was studied. The addition of Pluronic caused severe instabilities of the electrical current and the signal of the UV detector. This study reveals remarkable positive effect of a low pressure applied to the inlet buffer vial during the analysis, which apparently stabilizes the electrical current and the detector signal. The effect of hydrodynamic flow induced by the pressure applied on the separation efficiency was studied and the significance of this effect was discussed. Pluronic F-127, as a representative of synthetic macromolecules, was compared with dextran, as a representative of natural polymers, in terms of separation power, selectivity and repeatability of migration times.
Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.
- MeSH
- chromatografie kapalinová metody přístrojové vybavení MeSH
- čočka chemie MeSH
- financování organizované MeSH
- hmotnostní spektrometrie metody přístrojové vybavení MeSH
- lidé MeSH
- mikrofluidní analytické techniky MeSH
- molekulární sekvence - údaje MeSH
- proteiny analýza genetika MeSH
- rýže (rod) chemie MeSH
- semena rostlinná chemie MeSH
- testování materiálů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- hodnotící studie MeSH
