Twin trough glass chamber

n-Hexane extract (20 mg) of B. suffruticosa was dissolved in 40 ml of n-hexane, treated with a pinch of charcoal, filtered and the volume was made up to 50 ml in a volumetric flask. This sample solution was used for the thin layer chromatography (TLC) fingerprinting profile. TLC plates consisted of 10 cm × 10 cm, precoated with silica gel 60 F₂₅₄ TLC plates (E. Merck) (0.2 mm thickness) with aluminum sheet support. The spotting device was a Camag Linomat V Automatic Sample Spotter (Camag, Muttenz, Switzerland); the syringe, 100 µl (Hamilton). The developing chamber was a Camag glass twin trough chamber (20 cm × 10 cm); densitometer a Camag TLC Scanner 3 linked to winCATS software (Camag, Muttenz, Switzerland). The experimental conditions were kept constant where, the temperature was 25°C ± 2°C and relative humidity was 40%. TLC fingerprint was developed by applying 25 µl of n-hexane extract (100 mg/50 ml) in a duplicate along with standards, lupeol and β-sitosterol with band size of 8 mm, and distance between the tracks of 12 mm. Plate was developed in a solvent system of toluene: Methanol (9.3:0.7), dried and observed under ultraviolet (UV) 254 nm and UV 366 nm. The plate was derivatized with anisaldehyde-sulfuric acid reagent followed by heating at 100°C until the colored band appeared. The R_F value and color of the resolved bands were noted.

Thin layer chromatography (TLC) was carried out on precoated silica gel F254 plates (0.2 mm, Merck, Darmstadt, Germany) as stationary phase and ethyl acetate: Glacial acetic acid: Formic acid: Methanol (7.5:0.2:0.3:1, v/v) as mobile phase. Plates were derivatized using 1% DMAC in 3M hydrochloric acid[30 (link)] that is specific for procyanidins (+) catechin was used as standard. Antioxidant bioautography[31 (link)] using 0.1% DPPH as spray reagent was carried out. High-performance TLC (HPTLC) study was carried out for confirming the presence of (+) catechin and procyanidin B2 reported to be present in cocoa powder.[13 (link)] HPTLC fingerprinting was performed on precoated silica gel F254 plates at room temperature. Solutions of standards and sample were applied to the plates using the Camag (Muttenz, Switzerland) linomat V sample applicator equipped with a 100 μl Hamilton (USA) syringe. Ascending development to a distance of 80 mm was performed using the same mobile phase as that used in aforementioned TLC, in a Camag glass twin trough chamber saturated with mobile phase vapor for 25 min. After development, the plates were dried and then scanned using a Camag TLC scanner with WINCAT software (Camag, Switzerland).

The sample and standards were separately spotted as bands of width 5 mm with Camag microliter syringe on precoated silica gel aluminum plate 60 F₂₅₄ (20 cm × 10 cm with 0.2 mm thickness), using a Camag Linomat-V applicator (Camag, Switzerland). The input instructions regarding slit dimension (4 mm × 0.45 mm) and scanning speed (20 mm/s) were defined using win-CAT-V 1.2.3 software (Camag, Switzerland). The mobile phase consisted of toluene: ethyl acetate: formic acid: methanol (3:4:0.8:0.7, v/v/v). The plates were developed up to 85% of total TLC plate height in a horizontal Camag twin trough glass chamber (10 cm × 20 cm) which was saturated with the mobile phase (10 mL in each side) for 30 min at RT and relative humidity 60%. The TLC plate was dried in current air with the help of an air dryer. The densitometric scanning was performed using Camag TLC scanner of III in the absorbance mode at 254 nm using deuterium lamp source.