Development of a spectrophotometric method for the measurement of thiols at trace levels

Abstract

A spectrophotometric method is described for the quantitative measurement of aliphatic and aromatic thiols. The method is based on the derivatization of thiols with 5,5′-dithiobis(2-nitrobenzoic acid), commonly known as Ellman's reagent. This results in the simultaneous formation of mixed disulfides and a common product, namely 5-mercapto-2-nitrobenzoate (referred to as thiolate anion). The latter species has an absorption maximum at 420 nm and a correspondingly high molar absorptivity (ε) in aqueous media above 10 000 l mol^–1 cm^–1. The method developed was optimized with respect to reaction time, the pH of the buffer in which the reagent was dissolved and the reagent concentration. Linear calibration plots of absorbance of thiolate anion as a function of concentration over the range 0 to 41 µmol l^–1 were obtained for all thiols analysed. The limit of detection (S/N = 3) was 0.41 µmol l^–1. Five repeated analyses of a 16 µmol l^–1 solution of L-cysteine methyl ester gave a coefficient of variation of 6% with respect to the absorbance of the thiolate anion. The method was found to be well suited both for the measurement of mixtures of thiols and for the quantification of cysteine bound to polystyrene beads via a glycine ‘linker’ molecule.