Isotachophoresis

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Stadia during an ITP separation of a mix of two analytes.
White: leading electrolyte; gray: terminating electrolyte; hatched: the analytes

The "self-sharpening effect" in ITP: due to a difference in electrical field, an ion will move faster when it comes in the previous zone, and slower when it comes in the next zone. Therefore it will return to its "own" zone. Below the corresponding electrical field for each zone

Isotachophoresis (Greek: iso = equal, tachos = speed, phoresis = migration) is a technique in analytical chemistry used to separate charged particles. It is a further development of electrophoresis. It is a powerful separation technique using a discontinuous electrical field to create sharp boundaries between the sample constituents.

In conventional electrophoresis almost all the current is carried by the electrolytic buffer. The sample constituents migrate under influence of a homogeneous electrical field. The buffer determines the pH of the medium as well as the dissociation degree of the sample elements according to their pK values. The sample constituents migrate at different speeds and become diluted by diffusion. Preparation of the sample is often necessary to concentrate the sample elements before application.

In isotachophoresis the sample is introduced between a fast leading electrolyte and a slow terminating electrolyte. After application of an electric potential a low electrical field is created in the leading electrolyte and a high electrical field in the terminating electrolyte. The pH at sample level is determined by the counter-ion of the leading electrolyte that migrates in the opposite direction. In a first stage the sample constituents migrate at different speeds and start to separate from each other. The faster constituents will create a lower electrical field in the leading part of the sample zone and vice versa. Finally the constituents will completely separate from each other and concentrate at an equilibrium concentration, surrounded by sharp electrical field differences. Specific spacer or marker molecules are added to the sample to separate physically the sample constituents one is interested in.

Isotachophoresis shows its superiority to conventional separation techniques when the maximum resolution is achieved with the latter. The choice of the experimental parameters remains complex but is very rewarding when successful.