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Instrumentation[ edit ] Figure 1: Diagram of capillary electrophoresis system The instrumentation needed to perform capillary electrophoresis is relatively simple.
A basic schematic of a capillary electrophoresis system is shown in figure 1. The system's main components are a sample vial, source and destination vials, a capillary, electrodesa high voltage power supplya detector, and a data output and handling device.
The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution.
To introduce the sample, the capillary inlet is placed into a vial containing the sample. Sample is introduced into the capillary via capillary actionpressure, siphoning, or electrokinetically, and the capillary is then returned to the source vial.
The migration of the analytes is initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high-voltage power supply. In the most common mode of CE, all ions, positive or negative, are pulled through the capillary in the same direction by electroosmotic flow.
The analytes separate as they migrate due to their electrophoretic mobility, and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device such as an integrator or computer.
The data is then displayed as an electropherogram, which reports detector response as a function of time.
Separated chemical compounds appear as peaks with different retention times in an electropherogram. Jorgensen and Krynn DeArman Lukacs, who first demonstrated the capabilities of this technique.
Smith and coworkers, and provides extremely high sensitivity for the analysis of very small sample sizes. Despite the very small sample sizes typically only a few nanoliters of liquid are introduced into the capillaryhigh sensitivity and sharp peaks are achieved in part due to injection strategies that result in concentration of analytes into a narrow zone near the inlet of the capillary.
This is achieved in either pressure or electrokinetic injections simply by suspending the sample in a buffer of lower conductivity e. A process called field-amplified sample stacking a form of isotachophoresis results in concentration of analyte in a narrow zone at the boundary between the low-conductivity sample and the higher-conductivity running buffer.
To achieve greater sample throughput, instruments with arrays of capillaries are used to analyze many samples simultaneously. Such capillary array electrophoresis CAE instruments with 16 or 96 capillaries are used for medium- to high-throughput capillary DNA sequencing, and the inlet ends of the capillaries are arrayed spatially to accept samples directly from SBS-standard footprint well plates.
Certain aspects of the instrumentation such as detection are necessarily more complex than for a single-capillary system, but the fundamental principles of design and operation are similar to those shown in Figure 1.
Detection[ edit ] Separation by capillary electrophoresis can be detected by several detection devices. In these systems, a section of the capillary itself is used as the detection cell. The use of on-tube detection enables detection of separated analytes with no loss of resolution.
In general, capillaries used in capillary electrophoresis are coated with a polymer frequently polyimide or Teflon for increased flexibility.
The portion of the capillary used for UV detection, however, must be optically transparent. For polyimide-coated capillaries, a segment of the coating is typically burned or scraped off to provide a bare window several millimeters long.
This bare section of capillary can break easily, and capillaries with transparent coatings are available to increase the stability of the cell window. According to the Beer-Lambert lawthe sensitivity of the detector is proportional to the path length of the cell.
To improve the sensitivity, the path length can be increased, though this results in a loss of resolution. The capillary tube itself can be expanded at the detection point, creating a "bubble cell" with a longer path length or additional tubing can be added at the detection point as shown in figure 2.
Both of these methods, however, will decrease the resolution of the separation. When used with a UV absorbance detector, the wider cross-section of the analyte in the cell allows for an illuminating beam twice as large, which reduces shot noise by a factor of two.
Together these two factors increase the sensitivity of Agilent Technologies's Bubble Cell CE Detector six times over that of one using a straight capillary. This cell and its manufacture are described on page 62 of the June issue of the Hewlett-Packard Journal.
Techniques for increasing the pathlength of the capillary: This mode of detection offers high sensitivity and improved selectivity for these samples, but cannot be utilized for samples that do not fluoresce. Numerous labeling strategies are used to create fluorescent derivatives or conjugates of non-fluorescent molecules, including proteins and DNA.
The set-up for fluorescence detection in a capillary electrophoresis system can be complicated.Free Examples of Argumentative essay. Argumentative essay samples.
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