Commission Regulation (EEC) No 2568/91Show full title

[F1 5. REFERENCE METHOD: SEPARATION OF THE ALCOHOLIC COMPOUNDS BY BASIC THIN-LAYER CHROMATOGRAPHY (TLC) PLATE U.K.

Preparation of the basic thin layer chromatography plates. Immerse or dip the silica gel plates (4.5) about 4 cm in the 0,2 M ethanolic potassium hydroxide solution (4.4) for 10 seconds, then allow to dry in a fume cupboard for two hours and finally place in an oven at 100 °C for one hour.

Remove from the oven and keep in a calcium chloride desiccator (3.7) until required for use (plates treated in this way must be used within 15 days).

Place hexane/ethyl ether mixture (4.13) (Note 3) into the development chamber, to a depth of approximately 1 cm. Close the chamber with the appropriate cover and leave thus for at least half an hour, in a cool place, so that liquid-vapour equilibrium is established. Strips of filter paper dipping into the eluent may be placed on the internal surfaces of the chamber. This reduces developing time by approximately one-third and brings about more uniform and regular elution of the components.

Note 3: The developing mixture should be replaced for every test, in order to achieve perfectly reproducible elution conditions. Alternative solvent 50:50 (V/V) n-hexane/ethyl ether may be used. U.K.

Prepare an approximately 5 % solution of the unsaponifiable prepared in Part 1 in ethyl acetate (4.10) and, using the 100 μL microsyringe (3.3), depose 0,3 ml of the solution on a narrow and uniform streak on the lower end (2 cm) of the chromatographic plate (4.5). In line with the streak, place 2 to 3 μL of the material reference solution (4.11), so that the sterol, triterpene dialcohols and alcohols bands can be identified after developing.

Place the plate in the developing chamber (3.1). The ambient temperature should be maintained between 15 and 20 °C (Note 4). Immediately close the chamber with the cover and allow eluting until the solvent front reaches approximately 1 cm from the upper edge of the plate. Remove the plate from the developing chamber and evaporate the solvent in a flow of hot air or by leaving the plate for a short while, under a hood.

Note 4: Higher temperature could worsen the separation. U.K.

Spray the plate lightly and uniformly with the 2,7-dichlorofluorescein solution (4.12) and then leave to dry. When the plate is observed under ultraviolet lamp (3.2), the sterols, triterpene dialcohols and alcohols bands can be identified through being aligned with the spots obtained from the reference solution (4.11). Mark the limits of the bands along the edges of the fluorescence with a black pencil (see TLC plate in Figure 1).

By using a metal spatula, scrape off the silica gel of the marked area. Place the finely comminuted material removed into the filter funnel (3.4). Add 10 mL of hot ethyl acetate (4.10), mix carefully with the metal spatula and filter (under vacuum if necessary), collecting the filtrate in the conical flask (3.5.) attached to the filter funnel.

Wash the residue in the flask three times with ethyl ether (4.3) (approximately 10 mL each time), collecting the filtrate in the same flask attached to the funnel, evaporate the filtrate to a volume of 4 to 5 mL, transfer the residual solution to the previously weighed 10 mL test tube (3.6), evaporate to dryness by mild heating, in a gentle flow of nitrogen, make up again using a few drops of acetone (4.6), evaporate again to dryness. The residue contained in the test tube consists of the sterol and triterpene dialcohols or the alcohols and triterpenic alcohols fractions.]

Textual Amendments

F1 Substituted by Commission Implementing Regulation (EU) 2019/1604 of 27 September 2019 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis.