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

[F1 PART 2 U.K. SEPARATION OF THE ALCOHOLIC COMPOUNDS FRACTIONS

1. SCOPE U.K.

The unsaponifiable matter prepared in Part 1 is fractionated in the different alcoholic compounds, aliphatic alcohols, sterols and triterpenic dialcohols (erythrodiol and uvaol).

2. PRINCIPLE U.K.

The unsaponifiable matter can be fractionated using basic thin layer chromatography (reference method), revealed and the corresponding bands scratched and extracted. As an alternative method of separation, HPLC using a silica gel column and UV detector and the different fractions collected. The aliphatic and triterpenic alcohols as well as the sterol and triterpenic dialcohols are isolated together.

3. APPARATUS U.K.

The usual laboratory equipment and in particular the following:

3.1.

Complete apparatus for analysis by thin-layer chromatography using 20 × 20 cm glass plates.

3.2.

Ultraviolet lamp with a wavelength of 366 or 254 nm.

3.3.

Microsyringes, 100 μL and 500 μL.

3.4.

Cylindrical filter funnel with a G3 porous septum (porosity 15-40 μm) of diameter approximately 2 cm and a depth of 5 cm, suitable for filtration under vacuum with male ground-glass joint.

3.5.

Conical flask with ground-glass female joint, 50 mL which can be fitted to the filter funnel (3.4).

3.6.

Test tube with a tapering bottom and a sealing glass stopper, 10 mL.

3.7.

Calcium dichloride desiccator.

3.8.

HPLC system, consisting of:

3.8.1.

Binary pump.

3.8.2.

Manual or automatic injector equipped with 200 μL injection loop.

3.8.3.

In-line degasser.

3.8.4.

UV-VIS or IR detector.

3.9.

HPLC column (25 cm × 4 mm i.d.) with silica gel 60 (5 μm particle size).

3.10.

Syringe filter, 0,45 μm.

3.11.

Conical flask 25 mL.

4. REAGENTS U.K.

4.1.

Potassium hydroxide minimum titre 85 %.

4.2.

Potassium hydroxide ethanolic solution, approximately 2 M.

Dissolve 130 g of potassium hydroxide (4.1) with cooling in 200 ml of distilled water and then make up to one litre with ethanol (4.9). Keep the solution in well-stoppered dark glass bottles and stored maximum 2 days.

4.3.

Ethyl ether, for analysis quality.

4.4.

Potassium hydroxide ethanolic solution, approximately 0,2 M.

Dissolve 13 g of potassium hydroxide (4.1) in 20 ml of distilled water and make up to one litre with ethanol (4.9).

4.5.

Glass 20x20 plates coated with silica gel, without fluorescence indicator, thickness 0,25 mm (commercially available ready for use).

4.6.

Acetone, for chromatography quality.

4.7.

n-Hexane, for chromatography quality.

4.8.

Ethyl ether, for chromatography quality.

4.9.

Ethanol of analytical quality.

4.10.

Ethyl acetate of analytical quality.

4.11.

Reference solution for thin-layer chromatography: cholesterol, phytosterols, alcohols and Erythrodiol 5 % solution in Ethyl acetate (4.10).

4.12.

Solution of 2,7-dichlorofluorescein, 0,2 % in ethanolic solution. Make slightly basic by adding a few drops of 2 M alcoholic potassium hydroxide solution (4.2).

4.13.

n-Hexane (4.7)/ethyl ether (4.8) mixture 65:35 (V/V).

4.14.

HPLC mobile phase n-hexane (4.7)/ethyl ether (4.8) (1:1) (V/V).

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.

6. SEPARATION OF THE ALCOHOLIC FRACTION BY HPLC U.K.

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.

Note 5: Carefully control the pressure of the HPLC pump, the ethyl ether can increase the pressure, adjust the flow to keep the pressure under control.] U.K.

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.