Capillary Electrophoresis Basics and Facts
Electrophoresis in Capillary Tubing, Nucleotides, Drugs, Chemicals
Nov 12, 2009
Capillary electrophoresis (CE) resembles a hybrid of both polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography(HPLC). High-voltages of 500 V/cm or greater are generated within narrow-bore (20-200 micrometer) capillaries. The high voltages cause electroosmotic and electrophoretic movement of buffer solutions and ions, respectively, within the capillary tubing. Separation of many different kinds of molecular species including nucleotides, drugs and many other biochemicals is based upon differences in size, charge, and hydrophobicity.
Capillary Electrophoresis Basics, Facts, Features and Characteristics
Features of capillary electrophoresis are:
- powerful electric field strengths,
- sophisticated ultraviolet and laser detectors that permit the electropherogram to be plotted similar to a chromatogram,
- small sample amounts, and corresponding reagents,
- qualitative and quantitative analyses with high efficiency and precision,
- excellent automation and ease of use,
- applicable to a wide selection of many diverse analyses.
Capillary Electrophoresis Basics of Instrumentation
The basic capillary electrophoresis instrumentation consists of:
- fused-silica capillary tubes with an optical window,
- regulated high voltage power supply,
- two electrode units,
- two buffer reservoirs,
- ultraviolet (UV) detector.
The capillary electrophoresis procedure typically proceeds as follows:
- The capillary ends are placed in the appropriate, respective buffer reservoirs.
- The detector and optical viewing window are aligned.
- The capillary is filled with buffer.
- The sample is introduced by dipping the end of the capillary into the sample solution vial, and applying pressure, vacuum, or voltage.
- Next, the immersed capillary is elevated about 12 inches (25 cm) above the detector-side of the buffer reservoir.
- Everything is ready for the instrument to be turned on and run.
Photo 5 below shows an 8-channel capillary electrophoresis autosequencer that can process 96 independent samples in a 24-hour period. Capillary sequencers are rapid, user-friendly and can be set up in about 15 minutes. Analyses of trace files are done in real time, and the files can be automatically exported to in-house data base software. Capillary electrophores is excellent for small-scale experiments and optimization of procedures for high throughput devices.
Capillary Electrophoresis List of Basic Applications
The CE procedure is invaluable for:
- DNA sequencing and genotyping,
- oligonucleotide purity determination,
- siRNAs study,
- mRNA separations,
- drugs: identification, purification, binding to proteins, enantiomer detection and separation, kinetic profiles,
- dye analyses.
Capillary Electrophoresis Ink and Dye FBI Applications
Capillary electrophoresis (CE) has recently been used for ink analysis by the FBI for criminology investigations (see photo 2 below, click to enlarge). A few nanoliters of ink is injected into a silica capillary containing buffered solution. Voltage is applied to the capillary, to separate the ink's components, and a photodiode array detector displays the detected components as an ultraviolet-visible spectrum. This rapid process is automated, and results are stored electronically to establish an ink profile reference library. Non-dye, ink additives may serve as as additional, useful markers. These studies suggest useful applications for dyes related to food, textiles and ink-jets.
Sources
Beckman Coulter. 2009. Introduction to Capillary Electrophoresis. (Handbook). http://www.beckmancoulter.com/literature/Bioresearch/360643-CEPrimer1.pdf
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