Electrospray ionization mass spectrometry (ESI-MS)

Trypan blue samples treated with either free laccase in DI water (38 mg/L of Trypan blue and 250 mg/L laccase) or laccase in 0.5 M Fe₂Cl₃ solutions (38 mg/L of Trypan blue) were analyzed using a Thermo LCQ Fleet Ultimate 3000 LC-MS and Xcalibur software. Tests conducted with Fe₂Cl₃ solution were aimed at understanding the effect of iron ions on Trypan blue degradation by laccase, because Fe₂Cl₃ was used as a coagulant for zein sorbents (Sect. 2.2). The 0.5 M Fe₂Cl₃ solution concentration was selected because it was well above the concentration used to make zein sorbents (0.07 M), and it would hence allow clearly noticing any effect of iron on Trypan blue degradation. Samples were directly analyzed by ESI-MS, bypassing the liquid chromatography column. Trypan blue samples without laccase or Fe₂Cl₃ were also analyzed for comparison. All samples were diluted with DI water and analyzed at pH = 11 (adjusted with NaOH). This pH was used to ensure that laccase could remain active when sorbents were prepared at alkaline pH.