Fraxinus spp. (leaf)

High Performance Thin Layer Chromatographic Identification

Ash leaf (leaf) HPTLC ID - NP and PEG reagent, 366 nm Ash leaf (leaf) (Fraxinus excelsior or Fraxinus oxyphylla) Lane Assignments Lanes, from left to right (Track, Volume, Sample): 2 µL Ash leaf 1 (Ph. Eur. extraction) 4 µL Ash leaf 1 (Ph. Eur. extraction) 6 µL Ash leaf 1 (Ph. Eur. extraction) 2 µL Ash leaf 1 4 µL Ash leaf 1 6 µL Ash leaf 1 2 µL Rutin 2 µL Chlorogenic acid 8 µL Ash leaf 2 10 µL Ash leaf 2 12 µL Ash leaf 2 6 µL Ash leaf 3 8 µL Ash leaf 3 10 µL Ash leaf 3  Reference Sample(s) Reference: Dissolve 3.5 mg of rutin in 5 mL of methanol. Dissolve 2.5 mg of chlorogenic acid in 5 mL of methanol.  Stationary Phase Stationary phase, i.e. Silica gel 60, F254  Mobile Phase Ethyl acetate, formic acid, water 80:10:10 (v/v/v)  Sample Preparation Method Sample: Mix 1 g of powdered sample with 10mL of methanol and sonicate for 10 minutes, then centrifuge or filter the solutions and use the supernatants / filtrates as test solutions Derivatization reagent: 1.) NP reagent, Preparation: 1 g of natural products reagent in 200 mL ethyl acetate; 2.) PEG reagent, Preparation: 10 g of polyethylene glycol 400 in 200 mL dichloromethane, Use: Heat plate 3 min at 100°C, dip (time 0, speed 5) in NP reagent, dry and dip (time 0, speed 5) in PEG reagent.  Detection Method Saturated chamber; developing distance 70 mm from lower edge; relative humidity 33%  Other Notes Images presented in this entry are examples and are not intended to be used as basis for setting specifications for quality control purposes. System suitability test: Rutin: yellow fluorescent zone at Rf ~ 0.20; Chlorogenic acid: blue fluorescent zone at Rf ~ 0.36. Identification: Compare result with reference images. The fingerprint of the test solution is similar to that of the corresponding botanical reference sample. Additional weak zones may be present. The chromatogram of the test solution shows a yellow zone corresponding to reference rutin and a faint blue zone corresponding to chlorogenic acid. There is an intense bluish zone at Rf ~ 0.42 just above the zone corresponding to chlorogenic acid (yellow arrows). There is a faint dark blue zone at Rf ~ 0.83 (blue arrow). Source: HPTLC Association