The identification of glycosylated anthracene C-glycosides can be done using a combination of chemical and analytical techniques, considering the characteristic properties of these compounds. Here’s an approach:



1. Chemical Tests:

  • Borntrager's Test:
    • Principle: Anthracene derivatives typically undergo hydrolysis with dilute sulfuric acid or potassium hydroxide to form anthraquinones, which exhibit specific color changes.
    • Procedure:
      • Hydrolyze the sample by boiling with dilute sulfuric acid.
      • Extract with an organic solvent like ether.
      • Add 10% ammonium hydroxide to the organic extract.
      • Observation: A pink or red color in the alkaline layer indicates the presence of anthraquinones, suggesting the presence of anthracene glycosides.
  • Shinoda Test (for confirmation):
    • Mix a small amount of the sample with ethanol and add a few magnesium turnings followed by concentrated hydrochloric acid.
    • Observation: A pink to red color may be observed due to flavonoids, which can co-occur with anthracene glycosides.

2. Spectroscopic Methods:

  • UV-Visible Spectroscopy:

    • Anthracene glycosides show characteristic absorbance in the UV region.
    • The appearance of absorption maxima around 250-350 nm can be an indication of the anthracene chromophore.
  • Infrared Spectroscopy (IR):

    • IR can identify functional groups specific to glycosides.
    • C-O-C stretching from the glycosidic bond will show peaks around 1050-1150 cm⁻¹.
    • The anthracene ring system will also have distinctive aromatic C-H stretching and bending vibrations.
  • Nuclear Magnetic Resonance (NMR):

    • ¹H NMR: Signals from aromatic protons will be present in the region of 7-8 ppm.
    • The presence of sugar moieties can be confirmed by anomeric proton signals typically around 4.5-5.5 ppm.
    • ¹³C NMR: The anthracene carbons will have chemical shifts in the aromatic region (100-150 ppm), and the sugar carbons can be identified by their characteristic chemical shifts.

3. Chromatographic Techniques:

  • Thin-Layer Chromatography (TLC):
    • Prepare a TLC plate using silica gel as the stationary phase.
    • Develop the plate using a suitable mobile phase, such as ethyl acetate:water
      acid (8:1:1).
    • After development, spray with an appropriate reagent like p-anisaldehyde or sulfuric acid.
    • Heat the plate, and anthracene glycosides should appear as colored spots, typically orange or yellow.
  • High-Performance Liquid Chromatography (HPLC):
    • Reverse-phase HPLC can separate glycosides based on polarity.
    • The retention time, in combination with UV detection (around 250-350 nm), helps confirm the presence of anthracene C-glycosides.

These tests, in combination, provide a reliable identification of glycosylated anthracene C-glycosides, ensuring specificity and confirmation through both chemical reactions and spectral data.