In 1958, Frederick Sanger won the Nobel Prize in Chemistry for determining of the structure of insulin using 2,4-dinitroflourobenzene (DNFB). DNFB derivatized the N-terminus of peptides by adding a chromophoric “tag” which allowed for spectroscopic detection in liquid chromatography (LC) analysis of non-chromophoric peptides. We investigated the reductive electrochemistry of nitro-substituted aromatics to assess the feasibility of electrochemical detection in LC analysis of DNFB tagged amines, amino acids and peptides. Cyclic voltammetry (CV, glassy carbon working electrode and Ag/AgCl reference) of nitrobenzene (1) in three different aqueous buffers (pH 7, 4.5, 2) produced an irreversible reduction wave (Epc = –0.66, –0.58, and –0.53 V for pH 7.0, 4.5, and 2.0, resp.). Following reduction, a new reversible redox couple appears (Eo = –0.03, 0.11 and 0.16 V for pH 7.0, 4.5, and 2.0, resp.). The identity of the species for the new redox couple was determined by CV of possible reduction products: aniline, p-aminophenol and n-phenylhydroxylamine. We concluded that 1 was reduced to the n-phenylhydroxylamine (2) in a four electron, four proton process. The product 2 can be reversibly oxidized to nitrosobenzene (3) in a two electron, two proton process. We also ran CV on 2,4-dinitroaniline (4) in the different buffers and observed a similar irreversible reduction followed by formation of a new reversible redox couple, similar to nitrobenzene reduction. Instead of one reduction peak, we observe two reduction peaks corresponding to reduction of each nitro group and formation of two adjacent new reversible redox couples. Using computational chemistry we predict the nitro group ortho to the amine is being reduced before the reduction of the para nitro group. The feasibility of using dual electrode detection in series will be discussed in LC-EC analysis of Sanger derivatized amino acids.

Electrochemical reduction of aromatic nitro compounds: Strategies for LC-EC analysis of Sanger tagged analytes

251st National Meeting (2016)

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