Isotachophoresis as a preconcentration technique in capillary zone electrophoresis

Isotachophoresis (ITP) preconcentration is an attractive approach to improving capillary zone electrophoresis (CZE) sensitivity. The relationship between ITP-CZE sensitivity and injection volume was studied, and novel strategies were explored to improve ITP-CZE performance.;The relationship between injection volume of a dilute sample and its concentration at peak maximum in an ITP zone was investigated. The concentration at peak maximum was predicted to increase linearly with injection volume for a given leading electrolyte concentration, as long as the analyte mass injected was very small. At a specific higher concentration, defined by the concentration of the leading electrolyte, the peak begins to broaden and form a flat top, characteristic of a typical ITP zone. The linear relationship begins to fail at concentration much lower than the leading electrolyte concentration. The relationship between sensitivity of counter-flow ITP-CZE and injection volume was investigated using angiotensins as test analytes. The results confirmed that sensitivity is a nearly linear function of injection volume for dilute samples. In addition, counter-flow ITP-CZE on directly coupled columns of different diameters was investigated to increase the injection volume and lower the detection limits in CZE. A 2.5 nM solution could be detected easily from 8.3 muL of sample. Moreover, the concept of migration markers was introduced to provide qualitative information for ITP. 2-Chloropropionic, glyoxylic, and levulinic acids were evaluated as migration markers for isotachophoresis of ribonucleotides on Ucon-coated capillaries. These migration markers divided the ribonucleotides into three groups: triphosphate, diphosphate, and monophosphate.;An automated comprehensive ITP-CZE system was developed. The sample was focused in one capillary by ITP and injected repeatedly into another smaller diameter capillary for fast CZE separation. Since only small portions of the concentrated zones were sequentially injected for CZE separation, overloading was prevented. Moreover, the sensitivity was enhanced because all of the concentrated zones were analyzed and the results summed. A single detector (only for the CZE dimension) was required, and accurate timing for CZE injection was not necessary. The system was evaluated using a mixture of angiotensins. Under optimized conditions, a detection limit of approximately 5 nM could be easily achieved by injecting 10 muL of angiotensin solution.