The reaction of antioxidants with DPPH radical would reduce the concentration
of DPPH radical and lower the absorbance and then provide
a negative peak on a constant background signal, while for
those without antioxidants effects would not change the constant
background signal (Niederländer et al., 2008).
This on-line assay was performed by using the method introduced
by Koleva et al. (2000) and Niederländer et al. (2008) with slight
modifications as shown in Fig. 1. The extracts (20 μL) were injected
into an HPLC system. HPLC separation was carried out as described
in the previous section. HPLC eluated compounds arrived to a
T-junction, where the methanolic DPPH solution with the concentration
of 6 × 10−5 mol/L was delivered via another LC pump with a
flow rate of 0.8 mL/min. After the eluates mixed with DPPH solution
in a reaction coil (10 m × 0.254 mm i.d. PEEK tubing), the negative
peaks were measured at 517 nm by DAD.