Prior to derivatization, acceptor solution was added to the extracted sample until a final volume of 100 µL was reached to have constant starting volumes for derivatization and ensure comparability between samples.
The samples were derivatized following a procedure based on a diazotization and subsequent iodination reactions [16 (link)]. Into 100 μL of the extracted sample, 100 μL hydriodic acid (55%) and 200 μL sodium nitrite (50 g/L) were added and the samples were shaken for 20 min at 300 rpm, transforming the amine group of the aromatic amines into diazonium ions. To destroy the surplus of nitrite, 500 μL of sulfamic acid (50 g/L) was added, shaking subsequently for 45 min at 300 rpm. The samples were then heated in a water bath at 95 °C for 5 min to facilitate the substitution of the diazo group by iodine. To reduce the surplus of iodine, 250 µL of sodium sulfite (120 g/L) was added to the cooled down sample, which triggered an immediate discoloration of the initially brownish solution. Finally, 100 μL of alizarin red S (1% w/v) and 92 µL NaOH (10 M) were added to the samples to adjust the pH of the sample to 5.
The samples used for the optimization tests were derivatized automatically thanks to the PAL RTC from CTC Analytics AG (Zwingen, Switzerland). A few modifications were done to the procedure, such as vortexing the reagents before addition and the samples after reagent addition. For the method validation experiments, the derivatization was done manually due to the increased throughput needed, since with the PAL RTC only six samples could be derivatized at the same time, due to the six positions in the agitator.
The samples were derivatized following a procedure based on a diazotization and subsequent iodination reactions [16 (link)]. Into 100 μL of the extracted sample, 100 μL hydriodic acid (55%) and 200 μL sodium nitrite (50 g/L) were added and the samples were shaken for 20 min at 300 rpm, transforming the amine group of the aromatic amines into diazonium ions. To destroy the surplus of nitrite, 500 μL of sulfamic acid (50 g/L) was added, shaking subsequently for 45 min at 300 rpm. The samples were then heated in a water bath at 95 °C for 5 min to facilitate the substitution of the diazo group by iodine. To reduce the surplus of iodine, 250 µL of sodium sulfite (120 g/L) was added to the cooled down sample, which triggered an immediate discoloration of the initially brownish solution. Finally, 100 μL of alizarin red S (1% w/v) and 92 µL NaOH (10 M) were added to the samples to adjust the pH of the sample to 5.
The samples used for the optimization tests were derivatized automatically thanks to the PAL RTC from CTC Analytics AG (Zwingen, Switzerland). A few modifications were done to the procedure, such as vortexing the reagents before addition and the samples after reagent addition. For the method validation experiments, the derivatization was done manually due to the increased throughput needed, since with the PAL RTC only six samples could be derivatized at the same time, due to the six positions in the agitator.
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