Analysis of the Visual Spectrum of Methyl Orange in Solvents and in Hydrophobic Binding Sites

Abstract

The absorption curves of methyl orange (MO) and analogous p-aminophenylazobenzenes in organic and aqueous organic solvents are shown to consist of two severely overlapping bands. The curves have been resolved into two skewed component bands using a regression method. The blue shift of the absorption maximum obtained when organic solvents are added to aqueous solutions of MO, or when MO is bound to bovine serum albumin or a surfactant micelle, is the result of a change in relative intensities of the component bands. The low-frequency component is assigned to a π 1 → π 1 * transition of a solvate in which there is specific hydrogen-bonding interaction between solvent and the azo nitrogens, and the high-frequency component to a π 1 → π 1 * transition of a solvate in which the interaction is absent. The low-frequency component is favored by aqueous solvent compositions and by low temperatures. The free energies of interconversion of the species in various hydrogen-bonding solvents are correlated by the solvent surface tension but not by the dielectric constant. The results show that the shift in absorption maximum accompanying binding to a protein or micelle should be interpreted as a shift in an equilibrium rather than as a shift in transition energy.