Solution composition dependent variation in extinction coefficients for p-nitroaniline.

The dependence of the extinction coefficients for para-nitroaniline on solution composition has been investigated. The p-nitroaniline absorption spectrum is red-shifted with increasing ionic strength, with the consequence that the extinction coefficients at fixed wavelengths may vary significantly. The isosbestic wavelength for peptide p-nitroanilide/p-nitroaniline mixtures is similarly shifted. Poly(ethylene glycol) and bovine albumin, two additives frequently employed to eliminate enzyme loss from adsorption to cuvette and dilution vessel surfaces, also induce shifts in the p-nitroaniline spectrum. The use of a difference extinction coefficient at 381 nm, the p-nitroaniline maximum wavelength, is proposed to minimize the error resulting from solution composition dependent spectral shifts.