Principle and application of capillary gel electrophoresis

Capillary Electrophoresis (CE), also known as High Performance Capillary Electrophoresis (HPCE) or Capillary Separation (CESM), is a type of capillary separation channel driven by a high voltage DC electric field, depending on the components in the sample. A class of liquid phase separation technology that achieves separation by the difference in migration speed and distribution behavior, rapidly developed in the mid-to-late 1980s. It actually contains electrophoresis technology and chromatographic technology and its cross-over content. It is an analytical science relay high performance liquid chromatography. Another major development after that.

In 1987, Cohen published the work of capillary gel electrophoresis. When electrophoresis is moved from the gel plate to the capillary, a miraculous change occurs: the sensitivity of the analysis is increased to detect a change in one base, and the separation efficiency is up to one million theoretical plates; the analysis fragment can be small and small. To resolve the sequence of a single nucleotide, it is large enough to separate the DNA of Mb; the analysis time is reduced from the original hour to the minute and second. CE can be said to be the product of the perfect combination of classical electrophoresis technology and modern microcolumn separation technology.

one. Principle of capillary gel electrophoresis

The nature of the charge carried by different molecules is different, and the shape and size are different. In certain buffers or other solutions of electrolytes and PHs, the components in the sample migrate at a certain speed by the action of an electric field, thereby forming electrophoresis. The electrophoretic migration speed (v) can be expressed by the following formula:

v=uE
Where E is the electric field strength (E = V / L, V is the voltage, L is the total length of the capillary). u is the electrophoresis temperature.

Capillary gel electrophoresis is the electrophoresis of moving a gel on a plate into a capillary as a support. The gel is porous and functions like a molecular sieve, and the solute is separated one by one by molecular size. The viscosity of the gel is large, which can reduce the diffusion of the solute, and the peak shape is sharp, which can achieve the highest efficiency in CE. Joule heat is generated when current is passed through the conductor. The biggest limitation of traditional slab gel electrophoresis is that it cannot overcome the negative effects of Joule heat caused by high voltage at both ends. Joule heat can cause unevenness in temperature, viscosity and separation speed inside the screening medium, affecting migration, reducing efficiency, and widening the zone. Since this negative influence is proportional to the electric field strength, the introduction of high voltage is greatly limited. It is also difficult to increase the electrophoresis speed. Capillary electrophoresis separates the sample in a very fine column. The thin column can reduce the current and reduce the generation of Joule heat; at the same time, the heat dissipation area is increased, the heat dissipation efficiency is improved, the temperature difference between the center of the tube and the tube wall is greatly reduced, and various gradient differences in the radial direction of the column are reduced, thereby ensuring Efficient separation. Therefore, the electric field strength can be increased to 100 to 200 V/cm, and the separation quality can be improved comprehensively.

During the analysis, the capillary is filled with gel, and both ends of the capillary are subjected to high voltage, so that the charged molecules in the gel move to the opposite end of the capillary. Because the size of different molecules is different from the charge ratio, they move in the tube at different rates, and the end point of the capillary is also fast and slow. Capillary electrophoresis detects and separates different molecules accordingly.

two. Capillary gel electrophoresis

1. The required sample volume is small, the instrument is simple, and the operation is simple.

2. Fast analysis, high separation efficiency, high resolution and high sensitivity.

3. No need for nucleic acid dyes, safe and non-toxic.

4. No need to make glue, saving time and effort.

5. No need to apply glue to eliminate the error of manual analysis results.

6. Automatic results, including fragment size and sample concentration, the software can output electrophoresis peak map, gel electrophoresis map, DNA fragment base difference analysis, relative quantitative analysis.