4.4.6 Capillary Isoachophoresis (CITP)

Capillary isotachophoresis is a moving boundary technique using two buffer systems. The buffer systems trap the analyte zones and all zones lay in between “leading” and “terminating” electrolytes, where the leading electrolytes are those in the buffer with a higher EOF and the terminating electrolytes are those in the buffer with the lower EOF.

This bracketing affects the electric field experienced by each zone, creating different electric fields for each analyte. The electric field varies such that each analyte moves at the same velocity as that established by the leading electrolyte. Because velocity is defined as the product of the mobility and the electric field (velocity=mobility·field), this varying field acts to compliment the mobility such that all analytes move at the same velocity. Zones are formed and focused much in the same way as they are in CIEF; as soon as an analyte drifts away from its zone, it experiences a new electric field such that it either accelerates or decelerates until it migrates back to its zone and the steady state is reestablished.

According to the Kohlrausch Regulating Function, the concentration of analyte in a zone is determined by the concentration of the leading electrolyte. Analytes with a higher concentration than the leading electrolyte experience broadened until the concentrations match, whereas analytes with a lower concentration than the leading electrolyte sharpen. In this way, CITP can be used as a preconcentration step prior to other methods of analysis. Generally, up to 30-50% of the capillary can be filled with sample while still maintaining good separation and concentration.

Also due to the Kohlrausch Regulating function, increasing the concentration of the leading and/or trailing electrolytes can result in better separation and can improve detection of analytes that can be difficult to separate. However, when applying CITP, it can be difficult to identify a buffer system with leading and trailing electrolytes that have the appropriate mobilities while still maintaining the appropriate pH for the sample analysis. Additionally, only cations or anions can be analyzed by a given buffer system.