AbstractThe separation of enantiomeric forms of dansylated amino acids by isoelectric focusing in immobilized pH gradients (IPG) is demonstrated for the first time. Separations occur in a pH 3.0–4.0 IPG interval, in presence of 7murea, 10% methanol and 60 mM β‐cyclodextrin (CD) as chiral discriminator. It is found that the inclusion complex formed between the D‐form and CD has a lower pIthan the uncomplexed form (ΔpI= 0.05 for DL‐Phe and ΔpI= 0.025 for DL‐Trp); from this, it is calculated that the pKof the tertiary amino group in the dansyl moiety is lowered by 0.1 pH unit in the former case (D‐Phe) and by 0.05 in the case of D‐Trp (both values referring to 60 mM CD gels). For some racemates (e.g., DL‐Phe) the separation mechanism is still operative with CD concentrations as low as 20 mM. In our system 60 mM CD appears to be the solubility limit of CD. As the complex is stable in the electric field for at least 15 h, this separation mechanism could be exploited for purifying large quantities of pure D and L forms racemates in multicompartment electrolyzers with isoelectric Im