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EN
Free flow electrophoresis, or carrier-free electrophoresis, is a technique based upon the principle of mobility of chargeable species in the electric field. However, unlike more widespread gel electrophoresis, free flow electrophoresis does not utilise any kind of solid support medium. Instead, species to be separated move within the space filled with aqueous buffer, which is constantly pumped in the direction perpendicular to the direction of the applied electric field. Such set-up allows for continuous separation, as compounds of the sample being processed form separate bands and leave the separation system through several different outlets located along the edge of the chamber. Furthermore, the use of mild separation conditions increases the chance of biologically active compounds retaining their activity after the separation is finished. These qualities make free flow electrophoresis an excellent tool for protein research and cytology. In this review, the basic theoretical aspects of the technique are outlined, with a special emphasis placed on various modes in which free flow electrophoresis can be operated. Besides, a review of milestone papers related to free flow electrophoresis technology is presented. Apart from the devices following the original concept of Barrollier and Hannig, an insight into the construction of recirculating instrumentation, multicompartment electrolysers as well as radially-symmetric chambers is provided. A special focus is placed on patents and commercialized solutions. Finally, the challenges of scaling-down free flow electrophoresis to micro-dimensions are introduced.
EN
Influence of the following variables such as adsorbent type, type and concentration of organic modifier in mobile phase, type and concentration of ion-pairing reagent, or pH of the mobile phase buffer on retention of some synthetic peptides in reversed-phase high-performance thin-layer chromatography systems has been investigated. The investigations have been also focused on influence of the variables mentioned on solute zone shape regarding optimization of their separation. Remarks about solute retention mechanism have been also provided.
EN
High performance liquid chromatography (HPLC) is an instrumental analytical technique, which is widely used for a separation and determination of a mixture of components in many samples (e.g. of biomedical, pharmaceutical, food, and environmental origin). Despite several decades of the development of this technique, some aspects of the chromatographic process are still open to questions. This is particularly related to mechanisms of retention and selectivity of a separation. Improvement of the separation selectivity can be achieved by a change of the stationary phase type and qualitative and/or quantitative composition of the mobile phase. The replacement of the stationary phase does not ensure a smooth change of selectivity and retention, however, it generates additional costs of analysis. Therefore, the optimal conditions of chromatographic separation can be easily obtained by the change of a composition of the mobile phase, i.e. the type and/or concentration of its modifier (organic solvent). This paper presents an overview of approaches to explanation and interpretation of an influence of mobile phase composition on the retention and separation selectivity in liquid chromatography systems with particular emphasis on modifier type of eluent in the reversed phase high performance liquid chromatography (RP HPLC).
EN
Liquid chromatography and electrophoresis techniques are very often applied in contemporary laboratory practice. These techniques usually show different separation selectivity. It is due to various separation mechanisms involved in these two modes. In the former, partition of solutes between stationary and mobile phases influences on separation selectivity and retention contrary to the latter in which electrophoretic effect is involved in separation mechanism. The features mentioned are very useful for combination of these two techniques into two-dimensional separation of complicated samples of biomedical and environmental origin. Development of such approach is a very promising for contemporary separation sciences. The paper presents an overview of two-dimensional separation techniques, in which both liquid chromatography and electrophoresis have been involved especially in continuous mode.
EN
The study describes the solvent extraction procedure and improved version of TLC method for the determination of rhaponticin and deoxyrhaponticin in rhizomes and petioles of two species of Rheum L. genus. Extracts from underground parts and petioles of plant were separated on thin layers of silica using a mixture of dichloromethane-95% ethanol-methanol (8:1:1). The bands of rhaponticin and deoxyrhaponticin were satisfactorily separated from the remaining extract components permitting their densitometric quantitative and qualitative determination. The concentration of these compounds in Rheum undulatum was higher than in Rheum rhaponticum. The amount of rhaponticin and deoxyrhaponticin ranged between 0.3 and 31.5 mg/g.
6
Content available remote Elektrochromatografia planarna w układzie zamkniętym
EN
Planar electrochromatography in closed system (pressurized planar electrochromatography, PPEC) is the separation mode in which mobile phase is driven into movement by electroosmotic effect relative to adsorbent layer of the chromatographic plate. Solute band migration in the PPEC mode is governed by two effects: solute partition in a mobile phase - stationary phase system and electrophoresis when solutes undergo dissociation. Development of the methodology and equipment for PPEC are described in the paper. Attributes of the mode such as high separation efficiency and selectivity changes relative to liquid chromatography, especially thin-layer chromatography, are discussed. The mode is characterized by relatively short separation time and high performance of the separating system. Plate height approaches the value equal to two particle diameter of the stationary phase (adsorbent layer). Different separation selectivity mentioned is especially advantageous for application in two dimensional separation. The example of such separation of a test dye mixture, applying high performance thin layer chromatography in the first dimension and pressurized planar electrochromatography in the second one, is also presented and discussed in the paper. The mentioned advantages are very promising for development of the mode and its future application in laboratory practice. However, few main challenges concerned with production of chromatographic plates dedicated to the mode and construction of the equipment convenient for the operator are primary concern of future development of the mode.
EN
Fractions in which 37 organic compounds were identified by means of GC-MS method were isolated using extraction of four propolis samples and bud exudate fromPopu/usnigra. Six of them were identified in propolis for the first time. They are: 4-hydroxybutyric acid (4-hydroxybutanoic acid), 3-hydroxybutyric acid (3-hydroxybutanoic acid), phthalic acid, 4-hydroxyhydrocinnamic acid (phenyllactic acid), imidazole and phenetole. The main components of propolis fraction are acids: benzoic,cŤ- and trans-p-coumaric, ferulic, caffeic and cinnamic. The compounds were determined by HPLC technique (according to analysis conditions estimated with Dry-Lab software) applying calibration curve calculation for the standards of those acids. Propolis ethanolic extracts consisted of 3.5-8.8% of dry residue and 2.9% bud exudate extract fromPopu/us nigra. The percentage of acids in the isolated samples was determined in the range of 30% to 78%. The content of the compounds varied in different samples which confirms the fact that propolis composition is not constant. The determination of phenolic acids by HPLC technique in optimal conditions may be essential useful for propolis standardization.
PL
Metodą GC-MS zidentyfikowano 37 związków organicznych we frakcjach wyizolowanych po ekstrakcji czterech próbek propolisu oraz próbki wydzieliny pączków topoli Populus nigra. Sześć z tych związków zidentyfikowano w propolisie po raz pierwszy. Są to kwasy: 4-hydroksymasłowy, 3-hydroksymasłowy, ftalowy, 4-hydroksycynamonowy oraz imidazol i fenetol. Głównymi składnikami frakcj i propolisu są kwasy: benzoesowy, p-kumarowy, ferulowy, kawowy i cynamonowy. Związki te oznaczono metodą HPLC w układzie zoptymalizowanym za pomocą oprogramowania Dry-Lab. Ilościowy skład wyznaczono z krzywych kalibracyjnych związków wzorcowych chromatografowanych w tych samych warunkach jak badane próbki. Wyizolowane frakcje stanowiły od 3.5 do 8.8% suchej pozostałości etanolowego ekstraktu propolisu i 2.9% ekstraktu pączków topoli Populus nigra. W wyizolowanych frakcjach procent kwasów mieścił się w granicach od 30 do 78. Próbki różniły się zawartością ilościową poszczególnych kwasów, co potwierdza fakt, że skład propolisu niejest stały. Oznaczanie fenolokwasów metodąHPLC w optymalnych warunkach analizy może być przydatne do standaryzacji propolisu.
EN
Optimization of separation of eleven phenolic acids in reversed-phase high performance liquid chromatography (RP HPLC) systems by employing the Drylab software is presented. A gradient of a binary water-organic modifier (methanol, acetonitrile and tetrahydrofuran) mobile phase was applied. Fine tuning of chromatographic system conditions by the software was necessary to obtain good separation in all mobile phase systems. The simulated - and experimental retention data show good agreement. The optimized chromatographic system was applied to the separation of phenolic acids in a plant extract.
PL
Przedstawiono optymalizację rozdzielenia jedenastu fenolokwasów w odwróconym układzie faz przy wykorzystaniu oprogramowania Drylab. W układzie chromatograficznym zastosowano gradient dwuskładnikowej wodnoorganicznej fazy ruchomej. Podejście to umożliwiło precyzyjne wybranie warunków prowadzenia procesu chromatograficznego badanych substancji przy zastosowaniu wszystkich trzech użytych modyfikatorów (metanolu, acetonitrylu i tetrahydrofuranu). Symulowane wartości retencji i rozdzielenia wykazują dobrą zgodność z danymi eksperymentalnymi. Zoptymalizowane warunki prowadzenia procesu chromatograficznego zostały wykorzystane do przykładowego rozdzielania fenolokwasów w ekstrakcie roślinnym.
EN
An apparatus was constructed in which the eluate from micro-HPLC chromatograph (reversed-.phase (RP) system) is transferred directly in the form of an aerosol upon a moving silica TLC plate (normal-phase (NP), nonaqueous system) so that a two-dimen-sional chromatogram was obtained. The combination of RP and NP systems resulted in marked increase of separation efficiency owing to quite different selectivities of the two systems.
PL
Skonstruowano przyrząd (w którym eluat z kolumny mikro-HPLC (w układzie odwró-conym faz, RP-8-woda + metanol) jest nanoszony, w postaci aerozolu, bezpośrednio na linię startu przesuwającej się równolegle płytki z cienką warstwą żelu krzemionkowego. W rezultacie otrzymuje się chromatogram dwuwymiarowy. Połączenie układów RP i NP zapewnia znaczny wzrost ęfektywności rozdzielenia ze względu na odmienne charakterystyki selektywności obu tych układów.
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