Determination of ascorbic acid in citrullus lanatus red flesh juice by Thin Layer Chromatography (TLC) and Reverse Phase High Performance Liquid Chromatography (RP-HPLC) / Nurul Nabilah Akmal Hashim

Vitamin C or ascorbic acid is an essential water-soluble vitamin known for its antioxidant properties and act as a cofactor in numerous physiological reactions in the body. Being unable to be synthesized by human, ascorbic acid is consumed from most fruits and vegetables. Citrullus lanatus, a tropical fruit with green smooth hard rind and juicy flesh provides a rich source of ascorbic acid. For this study, Thin Layer Chromatography (TLC) and reverse phase High Performance Liquid Chromatography (RP-HPLC) separation methods were used to determine ascorbic acid in the red flesh of Citrullus lanatus. Ascorbic acid was extracted from Citrullus lanatus red flesh juice using 3 % orthophosphoric acid and 8 % acetic acid by solid-liquid extraction method. Qualitative estimation was carried out by TLC method using several solvent systems and the optimized solvent was applied as the mobile phase in RP-HPLC. As for quantitative analysis, RP-HPLC was performed on a ZORBAX Eclipse XDB-C18 analytical column (250 x 4.6 mm , 5µm) using isocratic elution mobile phase 0.1 % orthophosphoric acid and acetonitrile (95:5, v/v) with flow rate of 1 mL/min, column temperature of 25 ˚C, injection volume of 20 µl and the wavelength detection of 254 nm. The limits of detection (LOD) and quantification (LOQ) were found as 0.47 µg/mL and 1.43 µg/mL respectively. Ascorbic acid was detected by RP-HPLC separation method at retention time of 2.796 minutes. High selectivity and sensitivity of RP-HPLC separation method compared to TLC allows ascorbic acid in red flesh of Citrullus lanatus to be detected and quantitated.