The use of carrier ampholyte-free IEF (CAF-IEF) with ITP mobilization and conductivity detection in ITP mode for preconcentration and analysis of amino acids is demonstrated. The analytical procedure consists of three subsequent steps. In the first step, amino acids are continuously dosed from an infinite volume reservoir by electromigration to the column, where a sharp, stationary neutralization reaction boundary (NRB) is created in between acidic and basic primary electrolyte. Here, amino acids are selectively focused (trapped), if their pI falls to the pH difference on both sides of the NRB (pH gap). Amino acids create sharp rectangular zones, arranged according to their pI values. In the second step, focused zones are mobilized. After accumulation of the detectable amount of amino acids, dosing electrolyte in the infinite volume reservoir is changed for the mobilizing electrolyte. The migration mode is changed from CAF-IEF to ITP and substances start to migrate toward the analytical capillary. In the third step, analytes are transferred into the analytical column equipped with a conductivity detector and are detected in the new leading electrolyte in an ITP migration mode. The presented CAF-IEF-ITP-ITP with time-dependent accumulation of the large-volume sample enables to achieve in a reasonable time a 100 times lower c-LOD (here in orders of nmol/L), than can be reached by conventional hyphenated ITP-ITP.