Division 6 SEPARATION OF THE ALCOHOLIC FRACTION BY HPLCF1

The unsaponifiable obtained from Part 1 is dissolved in 3 mL of the mobile phase (4.14), filter the solution with a syringe filter (3.10) and reserve.

Inject 200 μL of the filtered unsaponifiable solution in the HPLC (3.8).

Run the HPLC separation at 0,8 mL/min, discard the first 5 min. and collect in 25 mL conical flasks (3.11) between the 5 and 10 min. for aliphatic and triterpenic alcohols and between 11 and 25 min for sterols and erythrodiol and uvaol (Note 5).

The separation can be monitored with an UV detector at 210 nm wavelengths or a refractive index detector (see Figure 6).

The fractions are evaporated until dryness and prepared for chromatographic analysis.