Characterization of the textile anthraquinone dye Reactive Blue 4

Analysis and characterization of the textile dye C.I. Reactive Blue 4 (RB4), a commercially important anthraquinone dye, as well as its hydrolysis products, were conducted by spectrophotometry, high performance liquid chromatography (HPLC), as well as advanced mass spectrometric techniques. Although spectrophotometric analysis can be used for the routine quantification of RB4 in aqueous solutions, HPLC analysis is necessary for the quantification of the unhydrolyzed, monohydroxy- and dihydroxy-RB4. The effect of both salt and base at concentrations typically used in reactive dyeing on the dye spectra was also investigated. Although moderate dye aggregation, leading to absorbance depression, was observed at relatively high dye and salt concentrations (1000 mg/l and 100 g/l, respectively), spectrophotometric and HPLC analysis was possible as long as the effect of salt and base was taken into account in the development of standard calibrations. Liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analyses led to the separation and identification of the parent RB4 molecule. LC/ESI-MS analysis of RB4 solutions led to the conclusive identification of the two RB4 hydrolysis products, as well as one impurity. Values of physico-chemical properties of RB4 were estimated using two software packages. The estimated RB4 LC50 value for acute toxicity to fish (1500 mg/l) agreed well with the acute toxicity EC5015 min value measured by the Microtox® procedure (1108 mg/l). Although the levels of RB4 in the environment are expected in the orders of magnitude lower than these LC50 and EC50 values, the effect of long-term, low-level dye exposure needs to be evaluated.