LC–MS-Guided CCD optimization and spectroscopic assignment of regioisomeric azo dyes BAO and BAM with TD-DFT-supported optical behavior

Abstract

A regioisomeric azo-coupling system based on diazotized 3-nitroaniline and 4-(dimethylamino)benzaldehyde was investigated to determine how reaction conditions control the formation of the two azo dyes BAO and BAM and how regioisomeric topology affects their structural and optical properties. The effects of acetic acid, aldehyde concentration, and temperature on the BAO/BAM ratio and total azo fraction were evaluated using a central composite design (CCD)-guided experimental design, while LC–MS enabled comparative quantification of reaction mixtures. Across the investigated design space, BAO was obtained as the major regioisomer in all runs, with BAO values of 76–84%, BAM values of 16–24%, BAO/BAM ratios of 3.167–5.250, and total azo fractions of 80–93%. The regioisomers were characterized by FTIR, NMR, GC–MS, and LC–MS, with gauge-independent atomic orbital (GIAO)-calculated chemical shifts supporting the NMR assignments. BAO exhibited moderate solvatochromism, whereas BAM showed a stronger solvatochromic behavior; both compounds displayed reversible acid/base switching for more than 20 cycles. Density functional theory (DFT), time-dependent density functional theory (TD-DFT), and protonation calculations supported the experimental observations, indicating that BAM is the more polarized regioisomer and exhibits greater sensitivity to solvent polarity and protonation. Overall, the study establishes correlations between reaction optimization, regioisomeric distribution, spectroscopic assignment, and medium-responsive optical behavior, providing insight into the influence of substitution topology on the structure–property relationships of azo chromophores.