Comprehensive isotachophoresis–capillary zone electrophoresis using directly inserted columns having different diameters with a periodic counterflow and dual ultraviolet detectors

Abstract

Isotachophoresis (ITP) coupled to capillary zone electrophoresis (CE) in a comprehensive manner was used to separate mixture components in both insufficient and sufficient quantities without heart-cutting or splitting. Examples of comprehensive ITP-CE involving multiple CE injections of preconcentrated ITP zones are demonstrated. In the comprehensive arrangement, all of the sample in the first dimension (ITP) is subjected to analysis in the second dimension (CE), without significant sample loss or decrease in sample detectability resulting from removal of a portion of the sample. This is especially important for analytes at low concentrations which may form a single mixed zone instead of individual ITP zones. Direct on-line coupling of ITP to CE in this comprehensive arrangement involved the use of columns having different diameters with one directly inserted inside of the other. A computer-controlled counterflow was applied when the isotachophoretic sample stack reached the bifurcation point. Large volume (10 μL) injections were made using an electrically insulated commercial polymeric rotary valve injector for increased reproducibility compared to previous comprehensive ITP-CE studies, with ITP and CE retention time RSD's ranging from 2 to 5%. An ultraviolet (UV) detector positioned at the bifurcation point was used to determine the beginning of CE injection. Application of a splitting voltage at the bifurcation point showed no affect on analyte transfer into the CE column. In using multiple injections of the ITP band(s), CE column overloading was not observed. © 2001 John Wiley & Sons, Inc. J Micro Sep 13: 361–370, 2001