Separation and Detection of Underivatized Amino Acids

Abstract

Amino acids (AA) are separated on a Hamilton PRP-1 polystyrene divinylbenzene column with a basic CH₃-CN:H₂O mobile phase containing a tetraalkylammonium (R₄N⁺) salt and a detector active counteranion. The underivatized AA are retained and separated as anions via a competing anion exchange process that occurs between the AA anion and the counteranion towards the R₄N⁺ salt, an equilibrium amount of which is maintained on the stationary phase according to the mobile phase conditions. An indirect detection (ID) is possible because of displacement of the detector active counteranion. Indirect photometric (IPD), fluorometric (IFD), or electrochemical (IED) detection is possible by using a counteranion that absorbs, fluoresces, or is electrochemically active. Parameters that influence retention and resolution are AA side chain structure and mobile phase pH. R₄N⁺ structure and concentration, organic modifier, and counteranion selectivity and concentration. These same parameters, in addition to the counteranion detector activity, also influence indirect detection. Detection limits for IPD are approximately 0.06 nmole (L-Ala) depending on the AA, mobile phase conditions, and counteranion chromophore for a 3:1 signal:noise ratio.