Chromatographic analysis of botanical based ascorbate
Aabid Khaliq Tantray, Sheikh Tajamul Islam
Three different stationary phases of column chromatography viz., neutral alumina (150 mesh), silica gel (230 mesh) and activated charcoal (Darco-G-60) were evaluated for ascorbate recovery efficiency from the methanolic extract of Emblica officinalis (Amla) fruit pulp. Screening of efficient mobile phase was carried using fifteen varied proportions of chloroform and methanol viz., 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:33, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65 and 30:70. The presence of ascorbate was detected by thin layer chromatography and purity confirmed by high performance liquid chromatography. Results were validated by performing a relative phytoascorbate recovery study. Maximum amount of ascorbate per gram dry weight (2.44%) was recovered when purification was followed by using neutral alumina as stationary phase, followed by silica gel (1.09%). Minimum recovery of ascorbate (0.85%) was observed when charcoal was used as stationary phase. Mobile phase in the ratio of 70:30 (Chloroform: Methanol) resulted in the maximum elution of ascorbate among all the proportions. Rf value of purified ascorbate was calculated as 0.667 against the standard (0.668) during TLC. In the HPLC study, the ascorbate peak in sample eluted within 3.73 minutes as compared to standard which eluted within 3.79 minutes.