Silica gel coated tlc plates

The reagents Luria–Bertani broth (Miller, Appleton, WI, USA) and Mueller–Hinton agar were procured from SRL Pvt. Ltd. (Mumbai, India), Himedia, whereas the ampicillin sodium injection was obtained from Saralife (New Delhi, India). Silica-gel-coated TLC plates, Wagner’s reagent, and Molisch reagent were procured from Merck, Darmstadt, Germany. All other reagents used were of analytical grade.

Silica gel-coated TLC plates (Merck, Darmstadt, Germany) were used for phytochemical analysis using various extracts of P. hysterophorus leaves prepared in water and ethyl acetate at room temperature (RT). The P. hysterophorus flowers extracts were prepared in methanol at high temperature (HT) and at RT in ethyl acetate and used on TLC [21 ]. The plates were dried and activated by heating at 110°C for 60 min. The extracts were dissolved in respective solvents. The extracts were applied 1.5 cm above from the bottom using glass capillary tubes. The solvent system acting as a mobile phase was allowed to run up to 7 cm from the point of application. The spots were visualized by exposure of plates to UV light and also by treatment with different developing reagents to ascertain the presence of various phytochemicals in the extracts. The retardation factor (Rf value) for each spot was calculated using the formula Rf = distance travelled by solute/distance travelled by solvent [26 ].

The sugar composition was analyzed by thin layer chromatography (TLC) using silica gel-coated TLC plates (Merck, Darmstadt, Germany). Glucose, galactose, mannose, fructose, xylose and rhamnose (Sigma Aldrich, St. Louis, MO, USA) at a concentration of 1 mg/mL were used as standards. Then, 30 µL of hydrolyzed EPS and standard solutions were transferred on TLC plates (Merck, Darmstadt, Germany) and dried. The plates were placed in a chromatographic chamber, which was first saturated by the developing phase containing 1-propanol:ethyl acetate:water 4:0.5:0.5 v/v for 2 h. Chromatograms were developed for 4 h; then, the plates were dried and sprinkled with 10% H₂SO₄ in ethanol and heated at 100 °C for 15 min to visualize separated compounds [49 (link)].