Spectrophotometric probing of the methyl red-bromine redox mechanism: a molecular mapping study with applications in grain and pulse analysis

Cysteine is a conditionally essential amino acid in the human body. Its sulfhydryl group plays a crucial role in key functions, including enzyme catalysis, protein structural stability, and the synthesis of the antioxidant glutathione. Thus, developing an analytical method where the sulfhydryl group directly participates in the determination process is highly effective. Here, a simple and sensitive spectrophotometric method is reported for the determination of cysteine based on the quantitative bleaching of methyl red dye (MR) by in situ generated bromine in an acidic medium. The method obeys Beer's law across a cysteine concentration range of 0-41 μM with a correlation coefficient of 0.99 and a relative standard deviation of 0.87% (n = 5). The proposed method was evaluated for interference from coexisting species using 33 μM of cysteine, and it was successfully applied to determine the natural cysteine content in soybeans and oats. Additionally, ground-state computational studies including molecular electrostatic potential mapping (MEPS), Mulliken charges, Natural population analysis (NPA), frontier molecular orbitals (HOMO-LUMO), and natural bond orbital (NBO) analysis were performed. These theoretical calculations confirm that bromine preferentially attacks the carboxyphenyl ring of the dye over the azo group, disrupting the intramolecular charge transfer (ICT) pathway and drives the observed spectral changes and bleaching mechanism.