This paper gives a comparison of the following methods used for separating mouse or rat spleen peritoneal cells, and mononuclear cells from human peripheral blood: adherence to glass wool, repeated adherence to polystyrene, adherence to polystyrene with subsequent detachment of adherent cells by scraping, or by lidocaine or dispase treatment, adherence to polystyrene coated with foetal calf serum; and subsequent release of adherent cells by means of EDTA, adherence to Sephadex G-10 columns, phagocytosis of carbonyl nickel and subsequent separation on the Ficoll-Verografin mixture, or phagocytosis of silica particles. The techniques in italics seem most suitable for routine use.
The paper provides information about the on-line monitoring of components analysed by capillary electrophoresis. For this purposes we developed a whole-capillary transverse scanning detection system, which helps to improve and control the separation processes. A picture from a colour line scanner was used as a source of basic information for autonomous control of the separation process by regulation of the high voltage source. The application and algorithms for machine vision were designed in the progressive graphic development system LabVIEW. Real-time control of the separation process was implemented in a compact control process logic controller. The performance of the detection system was evaluated and the function of the overall system was tested by performing isotachophoretic analysis of a model mixture.
- Publication type
- Journal Article MeSH
A review on dispersive effects and on peak broadening in electromigration separation methods (capillary electrophoresis and electrochromatography) is presented, mainly covering papers published between the beginning of 1997 and the beginning of 2000. Most attention is drawn to work dealing with nonlinear effects that cause anomalous electromigration dispersion in electrolyte systems with two or multiple coions. Further, topics cover the comparison of electroosmotic and pressure-driven modes in electrochromatography, dispersive effects due to nonhomogeneous velocity fields in packed electrochromatography columns, to nonuniform electroosmotic flow, to sorption of analytes (mainly proteins) at the column wall or the stationary phase, and due to the influence of the nonideal column geometry like coiling or irregularities in shape.
- MeSH
- Adsorption MeSH
- Chromatography, Micellar Electrokinetic Capillary methods MeSH
- Chromatography methods MeSH
- DNA chemistry isolation & purification MeSH
- Electrophoresis, Capillary methods MeSH
- Micelles MeSH
- Models, Molecular MeSH
- Proteins chemistry isolation & purification MeSH
- Models, Statistical MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- DNA MeSH
- Micelles MeSH
- Proteins MeSH
Separation methods are widely used to isolate humic substances (HSs), to fractionate them before further investigation, and to obtain information about their structure and properties. Among the chromatographic methods, techniques based on a size-exclusion effect appear to be most useful, as they allow us to relate elution data to the molecular mass distribution of HSs. The limitations of this approach are discussed in this review. Gas chromatography with mass spectrometric detection is typically used to identify the products of pyrolysis or thermochemolysis of HSs; this technique is considered most important in the structural investigation of HSs. Electrophoretic methods (especially capillary zone electrophoresis) provide detailed characterization of HSs, but it is very difficult to relate the electrophoretic data to any specific subfraction, structure or properties of HSs. The electrophoretic patterns are often called "fingerprints" and can potentially be used for the identification and classification of HSs. This is limited, however, by the great diversity of the procedures employed and by the low degree of harmonization--no data on reproducibility and between-laboratory comparability are available. The same holds true, to a certain degree, for most methods utilized for the characterization of HSs. Separation methods play an important role in the examination of the interactions of HSs with heavy metals and other chemical pollutants. They allow us to determine binding constants and other data necessary to predict the mobility of chemical pollutants in the environment.
- MeSH
- Electrophoresis methods MeSH
- Chromatography, Gel MeSH
- Humic Substances chemistry isolation & purification MeSH
- Gas Chromatography-Mass Spectrometry MeSH
- Spectrophotometry, Ultraviolet MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Humic Substances 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
- Chromatography, Micellar Electrokinetic Capillary methods MeSH
- Electrophoresis, Capillary methods MeSH
- Enzyme Assays methods MeSH
- Isoelectric Focusing methods MeSH
- Isotachophoresis methods MeSH
- Capillary Electrochromatography methods MeSH
- Kinetics MeSH
- Humans MeSH
- Miniaturization methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Chemical warfare agents and their degradation products represent a broad group of compounds with different chemical properties (polarity, volatility, thermostability, etc.). These chemicals often have to be detected and determined in complex matrices and therefore highly efficient separation techniques hyphenated to selective and sensitive detectors play an indispensable role. This review offers an overview of selected papers devoted to the title subject. It cannot be considered as a comprehensive literature compilation but should allow the reader to obtain an insight into the application of separation techniques in the important area of human protection and control of chemical weapons.
- MeSH
- Chemical Warfare Agents analysis isolation & purification MeSH
- Chemical Terrorism MeSH
- Chromatography, Liquid methods MeSH
- Chromatography, Gas methods MeSH
- Electrophoresis, Capillary methods MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Environmental Monitoring methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- Chemical Warfare Agents MeSH
BACKGROUND: Protooncogene CD117 is a cytokine receptor important for hematopoietic element development. Currently, we are not able to routinely separate a sufficient quantity of bone marrow CD117+ cells for experimental purposes. AIM: The aim of this study was to establish an immunomagnetic separation method for CD117+ hematopoietic stem cell isolation and to estimate the expression of chosen BCl-2 family protein members in these elements. MATERIAL AND METHODS: 120 samples of human and murine bone marrow were acquired using the magnetic separation system. The cells were stained for CD117, BCl-2, BAX, and CD33 by an indirect fluorescent immunocytochemistry. RESULTS: The flow cytometry analysis showed only 2.6% CD117+ cells from human as well as mouse bone marrow which is insufficient for further experiments. Cytospin was not good for morphologic characterization and immunophenotyping due to the fragility and destruction of the studied cells. Therefore, cell suspension staining was selected and by this method we found CD117 positivity in 70% of the mononuclerar (CD33 positive) elements in the case of chronic myeloid leukaemia. Labelling of the BCl-2 family in this case showed antiapoptotic BCl-2 expression in 80 %, proapoptotic BAX expression in approximately 5%. CONCLUSION: Our results show that CD117 immunomagnetic separation from bone marrow material is not acceptable for experimental purposes. They demonstrate that the only practical useful for the bone marrow cell examination (morphology and immunophenotype) is cell suspension staining which uncovers the distribution of both cytoplasmic proteins and surface antigens of immature blood elements.
- MeSH
- Hematopoietic Stem Cells chemistry classification MeSH
- Immunomagnetic Separation * MeSH
- Humans MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Proto-Oncogene Proteins c-kit analysis MeSH
- Flow Cytometry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Proto-Oncogene Proteins c-kit MeSH
In this short communication we report optimized procedures for the chiral separation of non-charged [6]helicene (1) and cationic derivative 1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide (2) using high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) methods. The possibility of using capillary electrophoresis (CE) was also tested. The satisfactory results were obtained with SFC, where the highly selective resolution of four enantiopure 1 and 2 helicenes was achieved in a single run within 5min. The semi-preparative procedure for the isolation of P and M enantiomers of compound 2, including circular dichroism data, is reported here for the first time. The results could be used in further separations and analytical applications targeting carbohelicenes vs. positively charged helicene derivatives.
- Keywords
- Capillary electrophoresis, Chirality, Helicene, High-performance liquid chromatography, P/M enantiomer, Supercritical fluid chromatography,
- MeSH
- Electrophoresis, Capillary methods MeSH
- Polycyclic Compounds chemistry isolation & purification MeSH
- Stereoisomerism MeSH
- Chromatography, Supercritical Fluid methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- helicenes MeSH Browser
- Polycyclic Compounds MeSH
The bacteriophage K1/420 is a member of genus Kayvirus that was extensively studied as an alternative treatment to combat bacterial infections caused by antibiotic-resistant Staphylococcus aureus strains. Despite the promise of phage therapy, the development of clinical applications of phages is facing regulatory and technical hurdles before it can receive acceptance in the Western World. Suitable simple and accurate diagnostic techniques to control the quality of the phage, which would satisfy the requirements of regulatory authorities are still being discussed. Here, we present the conditions for the simultaneous separation and detection of phage K1/420 and S. aureus by CZE and by CIEF were found, and the phage isoelectric point was determined to be 3.6. After removing the cell debris, the phage was successfully purified from the crude phage lysate and pre-concentrated by preparative isoelectric focusing. Its zone was localized by the positions of colored pI markers in the cellulose bed. The phage from the harvested zone had a decreased ability to infect its host. However, it was suitable for its separation, detection and identification by capillary electrophoretic methods, MALDI-TOF MS and electron microscopy.
- Keywords
- Capillary electrophoretic methods, Kayvirus, MALDI-TOF MS, Pre-concentration, Preparative IEF, Purification, Separation, Staphylococcus aureus,
- MeSH
- Isoelectric Focusing methods MeSH
- Humans MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Staphylococcus Phages classification isolation & purification physiology MeSH
- Staphylococcus aureus virology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Liquid chromatographic and electrophoretic methods applicable to the separation of collagen and its fragments are reviewed. Special attention is paid to the separation of both stabile and labile crosslinking elements. Identification procedures exploiting the mapping of either collagen alpha-chains or of cyanogen bromide fragments are discussed. These methods can be used for diagnosing inborn errors of collagen metabolism using bioptic or necroptic samples. Analysis of urinary hydroxyproline-containing peptides or the determination of peptidically bound pyridinoline is suitable for measuring the intensity of collagen metabolism.
- MeSH
- Amino Acids analysis MeSH
- Chromatography, Ion Exchange MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Collagen metabolism MeSH
- Collagen Diseases metabolism therapy MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Amino Acids MeSH
- Collagen MeSH
