High performance thin layer chromatography fingerprinting

Gallic acid was quantified in EP extract and fractions by HPTLC fingerprinting [30]. Briefly, test solutions of EP extract/fractions were prepared at a 2 mg/mL concentration in methanol. Gallic acid solution was prepared at a concentration of 100 μg/mL in methanol. HPTLC Silica gel 60 F254 plate (Merck Life Sciences Ltd Pvt, Mumbai) with a dimension of 20 cm x 10 cm served as the stationary phase. Mobile phase was prepared by saturating the 20 cm x 10 cm Twin trough chamber with 10 mL of Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2) for twenty minutes. Using the Camag Linomat 5 sample applicator, outfitted with liquid nitrogen tank, 10 μL per sample/standard was sprayed in the form of bands onto the HPTLC plate at a distance of 1.5 mm from the plate base. Sample solutions were loaded onto the sample applicator with a 100 μL HPTLC syringe (Camag Linomat syringe 695.0014, Hamilton Bonaduz, Schweiz). The plate was air dried and developed in the Twin trough chamber up to a distance of 80 mm from the point of application. Using Camag TLC Scanner 3, the HPTLC plate was scanned densitometrically under a scanning speed of 20 mm/s at a single wavelength of 280 nm. Subsequently, UV absorption spectrum of bands corresponding to gallic acid was examined at the same wavelength.