Separation possibilities of three-dimensional capillary electrophoresis

Separation possibilities of three-dimensional (3D) capillary electrophoresis (CE) were studied in this work. They were demonstrated using phthalic acid as a model analyte and human urine as a complex ionic model matrix. Complexity of the selected problem ordering from several facts, such as (i) analyte present on a trace concentration levels, (ii) analyte present in multicomponent matrix and (iii) analyte migrating in the region of electropherogram in which is naturally present the majority of ionizable organic compounds (i.e. potential interfering compounds). 3D tandem was created by (i) isotachophoresis (ITP) preseparation stage (first), (ii) ITP analytical stage (second), and (iii) capillary zone electrophoresis (CZE) analytical stage (third). Comparison of 2D and 3D CE employing two and three different CE stages, respectively, revealed considerably enhanced separation capability of the 3D CE system. Although no single ITP was sufficient for the effective sample pretreatment, the mutual combination of these two ITP steps do it. The proposed ITP tandem was based on the different migration pattern of two spacers–analyte–matrix constituents under different acid-base conditions (pH 3.1 vs. pH 4.5 in ITP1 and ITP2, respectively), that can be, generally, very effective tool for acidic compounds present in multicomponent ionic matrices. Besides the enhanced separation selectivity/sample clean-up, the 3D CE method kept benefits of the hydrodynamically closed separation system with enhanced sample loadability, such as excellent (i) reproducibility of the measurements and (ii) concentration detection limits. Hence, this study clearly demonstrated great potentialities of 3D CE in the solving even the most advanced separation problems as can be found in bioanalytical field.