TABLE 4.

HPTLC parameters for identification of black cohosh.

Parameters	Description
Stationary phase	20 × 10 cm glass plates Si 60 F254 (Merck)
SST	0.1 mg/ml of actein, isoferulic acid and cimifugin are individually prepared in methanol
Application volume	2 μL of References and test solution for identification and 20 µL of test solution for test for the presence of A. podocarpa, A. dahurica, and A. cimicifuga
Developing solvent	Toluene, ethyl formate, formic acid 50:30:20 (v/v/v)
Development	20 min saturation (with filter paper), 10 min conditioning at 0% relative humidity (with molecular sieve), 70 mm distance from lower edge, room temperature = 23–27°C
Derivatization reagent 1 (identification)	Sulfuric acid reagent: 20 ml of sulfuric acid was mixed with 180 ml of methanol. The plate is dipped (time: 0, speed:3) and then heated at 100 °C for 5 min
Derivatization reagent 2 (test for adulteration with A. dahurica)	Antimony trichloride reagent: 8 g of Antimony trichloride were mixed with 200 ml of chloroform and shaken until completely dissolved. The plates were dipped (time: 1s, speed: 3) into the solution and then heated at 120 °C for 10 min
Derivatization reagent 3 (test for adulteration with A. cimicifuga)	Boric acid, oxalic acid reagent: 4 g of boric acid and 5 g of oxalic acid were individually dissolved in 150 and 50 ml of ethanol absolute, respectively, and then shaken until completely clear. The solutions were combined before derivatization. The plates were dipped (time: 1s, speed: 3) into the solution and heated at 120 °C for 5 min
Documentation	White light, UV 254 nm and UV 366 nm prior to derivatization, UV 366 nm and white light after derivatization