2-Acetyldimedone and 12 related compounds were employed as UV-active pre-column derivatizing agents for amino acids. Direct enantioseparation of the products was achieved using chiral anion exchanger stationary phases in polar-organic mobile phase mode. Under basic conditions, the reagents´ cyclic β-tricarbonyl motifs can give rise to exo- and endocyclic enols through tautomerization. However, with primary amines (proteinogenic and unusual amino acids, aminosulfonic and aminophosphonic acids), we exclusively observed the formation of exocyclic enamine-type products. Reaction yields depended strongly on the 2-acyl modification of the reagent; in particular, we observed a significant decrease when electronegative or sterically demanding substituents were present in α-position to the exocyclic carbonyl group. In addition to improving UV detectability of the products, the introduction of this protective group facilitated successful enantiomer separations of the amino acid derivatives on Cinchona-based chiral anion exchangers. Particularly high enantiomer selectivity was observed in combination with stationary phases bearing a new variation of selectors with π-acidic (electron-poor) bis(trifluoromethyl)phenyl groups. No racemization of the analytes occurred at any stage of the analytical method including the deprotection, which was achieved with hydrazine.