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Miniaturyzacja w wysokosprawnej elektroforezie kapilarnej

Mrass A., Bald E.

Wiadomości Chemiczne

2002

[Z] 56, 11-12

931-952

During the last decade high performance capillary electrophoresis (HPCE) has developed into a powerful analytical method. Despite the overwhelming advantages of this technique, it has not yet found widespread use in molecular biology labs in comparison with the slab gel electrophoresis. The main reason for the low acceptance of this method is performing separation in a single capillary which is analogous to using only one lane on a slab gel; despite the high speed of the separation, the overall throughput is still low. To comply with the current needs for fast and high throughput analysis, especially in the field of DNA sequencing, two approaches have been introduced: a multicapillary scheme called capillary array electrophoresis and a multichannel system etched into the surface of microchips. Capillary array electrophoresis (CAE) was first introduced in 1992. The search for a comprehensive instrument was achieved by several groups seeking an instrument capable of fast, automated, sensitive and most important of all, rugged operation. In CAE electrokinetic injection is the most popular method for sample loading. However adopting this mode of injection for DNA sequencing requires a thorough sample clean-up. To inject unpurified samples a so-called ‘base-stacking’ method has been described. One of the major problem in CAE is detection as it has to meet plenty of demands, among which the most important are: high speed, high sensitivity and high spatial resolution. Numerous approaches have been developed to address these challenges using either scanning or imaging technologies. Capillary electrophoresis (CE) on microchips is based upon microfabrication techniques developed in the semiconductor industry. The design of microchips for CE has undergone significant development from simple single-channel structures to increasingly complex ones capable of proceeding various analytical steps. Sensitive detection schemes are essential in microfabriacted devices in CE due to extremely small size of the detection cell. Laser induced fluorescence (LIF) is so far the most popular for this purpose. Other methods successfully coupled to microchips include mass spectrometry, electrochemical detection, Raman spectroscopy and holographic refractive index detection.

Metody zatężania analitów w kapilarze w wysokosprawnej elektroforezie kapilarnej

Mrass A., Bald E.

Wiadomości Chemiczne

2001

[Z] 55, 9-10

933-953

High performance capillary electrophoresis (HPCE) was born of the marriage of the powerful separation mechanisms of electrophoresis with the instrumentation and automation concepts of chromatography and it has developed into an exciting and extremely powerful analytical technique in recent years. Numerous advantages, such as rapid analysis times, high separation efficiencies, low consumption of chemicals and simplicity of the instrument makes HPCE competitive to other separation techniques. One of the greatest advantages is its diverse application range, mainly due to a numerous modes of separation. Originally considered primarily for the analysis of biological macromolecules, it has proved useful for separations of compounds such as amino acids, vitamins, pesticides, inorganic ions, organic acids, dyes, surfactants, peptides and proteins, carbohydrate, oligonucleotides and DNA restriction fragments, and even whole cells and virus particles. Moreover HPCE makes the separation of chiral compounds possible. However one restraint of the HPCE techniques is the low concentration sensitivity as a consequence of limited optical pathlength for directed on-capillary photometric detection (the most popular HPCE mode of detection) and the limited volume of sample solution that can be injected into the capillary. Therefore one of the greatest challenges in the area of HPCE methods development is to lower limits of detection (LLD) which is at least an order of magnitude higher than in high performance liquid chromatography (HPLC). Several on-line strategies have been demonstrated to decrease LLD in HPCE, like introducing detector cell with extended pathlenth, powerful detectors i.e., laser-induced fluorescence, on-line preconcentrators with the use of solid-phase extraction materials, coated/impregnated membranes or ion-exchange flow injection system coupled to capillary electrophoresis. Among different remedies, on-capillary concentration techniques are easy to apply and do not require alteration of HPCE instrumentation, which are isotachophoretic preconcentration, sample stacking ( originally developed for ionic compounds, and recently introduced for micellar electrokinetic chromatography (MEKC) sweeping technique. Mechanisms of concentration in diverse techniques are different. The concentration effect in isotachophoresis (ITP) relies on the adjustment of the concentration of the dilute sample to the higher concentration of the leading electrolyte. Preconcentration by ITP can be performed online or in coupled columns. In sample stacking, the concentration effect relies on the difference in electrophoretic velocities between the high- and low-conductivity zones (background electrolyte and sample zones, respectively). Sample stacking technique can be performed in CZE as well as in MEKC mode. From 10 to more than 1000-fold improvements in detector response have been documented. Sweeping , a new concept in MEKC, is defined as the picking and accumulating of analyte molecules by pseudostationary phase that enters and fills the sample zone upon application of voltage. As on-capillary concentration technique, it has provided more than 5000-fold increases in detection sensitivity. Recently developed a cation-selective exhaustive injection and sweeping technique improves peak heights of cation analytes a million-fold.