2.6. Thin-layer chromatography (TLC)
TLC was performed using aluminium silica gel 60 F254 plates (4 cm × 6.6 cm). Five microliters of each sample or commercial 5-CQA (used as the control) diluted in methanol, were spotted at 0.6 cm from the bottom of the plate. The TLC plate was then placed in a developing chamber containing butanol/water/acetic acid (6:2:2, v/v/v) at room temperature (Lewis et al., 1998 and Lu et al., 2004). When the solvent reached 0.4 cm from the top of the plate, the plate was removed, dried for 5 s with a hair-dryer, observed at 254 and 360 nm, and finally revealed using iodine. The retention factor (Rf) was calculated as the distance travelled by the individual compound, divided by the distance travelled by the solvent. Assays were performed in duplicate