Capillary isotachophoresis (CITP) is a specialized electrophoretic separation technique that employs a discontinuous buffer system consisting of a leading electrolyte (LE) and a terminating electrolyte (TE) to focus charged analytes into sharply defined, contiguous zones. All analyte zones migrate at the same velocity, determined by the leading ion, a condition known as the isotachophoretic state. CITP offers unique capabilities for sample concentration, purification, and preconcentration prior to downstream analysis, and is particularly valuable for trace enrichment of ionic analytes from dilute samples. Unlike zone electrophoresis, where analytes separate into discrete zones that broaden over time, isotachophoresis compresses and maintains narrow zones throughout the separation, yielding high local analyte concentrations that improve detection sensitivity.
The Discontinuous Buffer System
The fundamental requirement for isotachophoresis is the use of two buffer systems that share a common counterion but differ in their effective mobility. The leading electrolyte contains an ion with the highest effective mobility among all ionic species in the system, while the terminating electrolyte contains an ion with the lowest effective mobility. The sample is introduced between these two electrolytes. When the electric field is applied, the leading ion moves fastest and establishes the migration velocity for the entire system. Ions with intermediate mobilities arrange themselves in order of decreasing mobility between the leading and terminating zones. All zones then migrate at the same velocity, governed by the leading ion, and remain in contact with one another without gaps. The concentration of each analyte adjusts automatically according to the Kohlrausch regulating function, which dictates that the product of concentration and mobility is constant within each zone under steady-state conditions.
Focusing Mechanism and Zone Sharpening
The self-sharpening effect is the defining characteristic of isotachophoresis. If an analyte ion diffuses into a neighboring zone where the electric field differs, it experiences a force that returns it to its own zone. Ions that stray into a zone of higher field strength accelerate until they re-enter their correct zone, while those entering a zone of lower field strength decelerate. This dynamic focusing counteracts diffusion and produces exceptionally sharp zone boundaries. The concentration of analyte within each zone is determined by the concentration and mobility of the leading ion, not by the initial sample concentration. This unique property allows CITP to concentrate analytes by factors of 100 to 1000 or more, making it a powerful tool for trace analysis.
Instrumentation and Practical Considerations
CITP can be performed in the same capillary electrophoresis instrumentation used for other separation modes, provided the system can accommodate the discontinuous buffer configuration. A fused-silica capillary is filled with the leading electrolyte, followed by injection of the sample and then introduction of the terminating electrolyte at the inlet reservoir. A constant current power supply is typically preferred over constant voltage operation because the current remains stable during the separation, whereas the voltage changes as zones of different conductivity pass through the capillary. Detection is commonly performed using conductivity detectors, which respond to changes in zone conductivity, or UV-Vis absorbance detectors positioned at the capillary outlet. The formation of stable, reproducible zones requires careful selection of the leading and terminating electrolyte composition, including the choice of counterion, pH, and the addition of complexing agents or organic solvents to adjust selectivity.
Coupling with Capillary Zone Electrophoresis
One of the most important applications of CITP is as an on-line preconcentration step prior to capillary zone electrophoresis (CZE). In a coupled CITP-CZE configuration, the first segment of the capillary is used for isotachophoretic focusing of a large sample volume, while the second segment performs zone electrophoretic separation of the focused analyte bands. A conductivity or voltage switch is used to transfer the stacked zones from the CITP stage into the CZE stage. This approach can enhance detection sensitivity by two to three orders of magnitude compared to conventional CZE injection, enabling detection of trace components at concentrations in the nanomolar range. The hyphenated CITP-CZE technique has been applied to the analysis of peptides, proteins, nucleotides, pharmaceutical impurities, environmental contaminants, and food additives.
Applications in Bioanalysis
CITP has found extensive use in the bioanalytical field for the preconcentration and cleanup of complex biological samples. In proteomics, CITP is employed to concentrate dilute protein digests and remove high-abundance salts and buffers that would interfere with mass spectrometric detection. The technique has been integrated with capillary electrophoresis-mass spectrometry (CE-MS) workflows to improve the detection of low-abundance peptides and post-translational modifications. In clinical chemistry, CITP is used for the determination of organic acids in urine, monitoring of drug metabolites in plasma, and screening for inborn errors of metabolism. The ability to process large injection volumes without loss of resolution makes CITP particularly suitable for samples where the analytes of interest are present at trace levels within a complex matrix.
Current Trends and Future Directions
Recent developments in CITP include miniaturization on microfluidic chips, where isotachophoretic focusing is used to concentrate analytes in microchannel networks for integration with lab-on-a-chip devices. Advances in electrolyte design, including the use of zwitterionic buffers and non-aqueous solvent systems, have expanded the range of analytes amenable to isotachophoretic analysis. Computational modeling of the isotachophoretic process now enables rational optimization of buffer compositions and separation conditions. CITP continues to evolve as a complementary technique within the broader capillary electrophoresis toolkit, valued for its unique concentrating power and its ability to handle challenging sample matrices encountered in environmental monitoring, food safety, pharmaceutical development, and clinical diagnostics.
