The use of transient moving chemical reaction boundary (tMCRB) was investigated for the on-line preconcentration of native amino acids in heart-cutting 2D-CE with multiple detection points using contactless conductivity detection The tMCRB focusing was obtained by using ammonium formate (pH 8 56) as sample matrix and acetic acid (pH 2.3) as a BCE in the first dimension of the heart-cutting 2D-CE Different experimental parameters such as the injected volume and the concentration in ammonium formate were optimized for improving the sensitivity of detection A stacked fraction from the first dimension was selected, Isolated in the capillary, and then separated in the second dimension in the presence of a chiral selector ((+)-(18-crown-6)-2,3,11,12-tetra-carboxylic acid). This on-line tMCRB preconcentration coupled with heart-cutting 2D-CE was applied with success to the chiral separation of D,L-phenylalanine, and h,L-threonine in a mixture of 22 native ammo acids. The sample mixture was diluted in 0 8 M of ammonium formate, and injected at a concentration of 2 5 mu M for each enantiomer with a volume corresponding to 10% of the total capillary volume An LOD (S/N = 3) of 2 mu M was determined for L-threonine