Dual Electrode Detection in LC‐EC Analysis of Sanger‐tagged Amino Acids: Electrochemical Reduction of Aromatic Nitro Groups

Abstract

Analysis of amino acids is crucial to protein structure elucidation. Amino acids, amenable to separation by liquid chromatography (LC), can be detected by sensitive detectors used in liquid chromatograph including absorbance, fluorescence or electrochemical detector when derivatized. Derivatization of amino acids using suitable reagents enhances the separation and detection of amino acids using liquid chromatography. Sanger's reagent (1-fluoro-2,4-dinitrobenzene, DNFB) makes amino acids suitable for absorbance detection. However, little has been done in terms of liquid chromatography with electrochemical detection (LC-EC) of the DNFB derivatized amino acids. Furthermore, sensor development based on electrochemistry of nitroaromatics has increased dramatically for explosive detection. Since nitroaromatics are electrochemically active, it is essential to determine the reduction pathway. Cyclic voltammetry (CV), rotating disk electrode (RDE) and rotating ring disk electrode (RRDE) experiments have been performed on nitrobenzene (NB) and N-methyl-2,4-dinitroaniline (NMDNA). The results showed that the nitro group was reduced to hydroxylamine in aqueous solutions by addition of four electrons and four protons per nitro group and the hydroxylamine can be reversibly oxidized into a nitroso by removal of two electrons and two protons. Electrochemical detection of the nitro groups is appropriate for dual electrode detection of DNFB labeled amino acids in which reduction can occur on an upstream electrode and corresponding oxidation of the reduction product occurs on the downstream electrode.