Separation of acetylated amino acids

Acetylated amino acids (Nac-AA) are separated as anions on a reversed stationary phase from a mobile phase containing a quaternary ammonium (R₄N⁺) salt as a mobile phase additive. If the counteranion accompanying the R₄N⁺ or ionic strength salt is detector active than the separated NAc-AA derivatives can be detected by an indirect detection strategy. Variables influencing the separations are NAc-AA side chain structure and the mobile phase parameters such as hdyrophobicity of the alkyl groups in the R₄N⁺ salt, R₄N⁺ salt concentration, counteranion eluent strength, counteranion concentration, solvent composition, and pH. Indirect detection is influenced by these same mobile phase parameters as well as the properties of the detector active counteranion. The detection limit for indirect photometric detection at 287 nm using a tetrapentylammonium salt with a disodium 1,5-naphthalenedisulfonate—sodium benzoate counteranion mixture was about 70 pmol of NAc-AA depending on the amino acid injected as a 10-μ1 sample.