Strategies for continuous on-line high performance liquid chromatography coupled with diode array detection and electrospray tandem mass spectrometry for process monitoring of sulphonated azo dyes and their intermediates in anaerobic-aerobic bioreactors

On-line HPLC with diode array detection (DAD) coupled to electrospray tandem mass spectrometry (ESI-MS/MS) is presented as an integrated quality control and process integrated optimisation tool for the continuous monitoring of sulphonated azo dyes (SADs) and their intermediates in anaerobic and aerobic bioprocesses. Ion-pair RP-HPLC is found out to be more suitable for simultaneous monitoring of aromatic amines (AAs), sulphonated aromatic amines (SAAs) and sulphonated azo dyes in comparison to RP-HPLC with polar embedded phases. Monitoring of the anaerobic degradation of the diazo Reactive Black 5 displays the dependency of a two stage azo group reduction mechanism on the redox potential of the bioreactor. An autoxidation sensitive intermediate released from the anaerobic reduction is characterised by ESI-MS/MS for the first time. The functionality of the method is demonstrated by the control and evaluation of selective adaptation of bacteria to certain pollutants and the identification of unknown intermediates causing re-gaining colour released from azo dye treatment.