Experimental and theoretical study of the spectral behavior of Trypan Blue in various solvents

The absorption spectra of Trypan Blue were recorded in various solvents. The absorption maxima of the dye were found to be dependent on solvent polarity. NMR and IR data suggest that dye exists in the keto form in all solvents studied. Aggregation of Trypan Blue was observed at low pH values. The electronic absorption spectra of Trypan Blue in various solvents were also analyzed in terms of absorption wavelength and the refractive index value given as a function of f(n²) and a linear relationship was found. In a mixture of binary solvents (water and DMSO), the dye molecule tends to form a hydrogen-bonding solvated complex with the more polar solvent. The value of molecular complex formation constant (K_f) for the dye/DMSO complex and the Gibbs free energy (ΔG) were calculated. Density functional calculations indicate significant enhancement in internal hydrogen bonding for the keto form, resulting in a large energy difference between the keto and enol forms, consistent with the experimental observation of the existence of the keto form only. Time dependent density functional theory calculations suggest that the two visible absorption maxima observed experimentally arise from a single species in solution. The lower energy band is assigned to the HOMO → LUMO transition, and the second band to the HOMO → LUMO + 1 transition.