[ANNEX V U.K. DETERMINATION OF THE COMPOSITION AND CONTENT OF STEROLS AND TRITERPENES DIALCOHOLS BY CAPILLARY-COLUMN GAS CHROMATOGRAPHY
4. REAGENTS U.K.
4.1. Potassium hydroxide minimum titre 85 %. U.K.
4.2. Potassium hydroxide ethanolic solution, approximately 2 N. U.K.
Dissolve 130 g of potassium hydroxide (point 4.1) with cooling in 200 ml of distilled water and then make up to one litre with ethanol (point 4.10). Keep the solution in well-stoppered dark glass bottles and stored maximum two days.
4.3. Ethyl ether, for analysis quality. U.K.
4.4. Potassium hydroxide ethanolic solution, approximately 0,2 N. U.K.
Dissolve 13 g of potassium hydroxide (point 4.1) in 20 ml of distilled water and make up to one litre with ethanol (point 4.10).
4.5. Anhydrous sodium sulphate, for analysis quality. U.K.
4.6. Glass plates (20x20 cm) coated with silica gel, without fluorescence indicator, thickness 0,25 mm (commercially available ready for use). U.K.
4.7. Toluene, for chromatography quality. U.K.
4.8. Acetone, for chromatography quality. U.K.
4.9. n-Hexane, for chromatography quality. U.K.
4.10. Ethyl ether, for chromatography quality. U.K.
4.11. Ethanol of analytical quality. U.K.
4.12. Ethyl acetate of analytical quality. U.K.
4.13. Reference solution for thin-layer chromatography: cholesterol or phytosterols, and erythrodiol 5 % solution in ethyl acetate (point 4.11). U.K.
4.14. 2,7-dichlorofluorescein, 0,2 % in ethanolic solution. Make slightly basic by adding a few drops of 2 N alcoholic potassium hydroxide solution (point 4.2). U.K.
4.15. Anhydrous pyridine, for chromatography quality (see Note 5). U.K.
4.16. Hexamethyl disilazane of analytical quality. U.K.
4.17. Trimethylchlorosilane of analytical quality. U.K.
4.18. Sample solutions of sterol trimethylsilyl ethers. U.K.
To be prepared at the time of use from sterols and erythrodiol obtained from oils containing them.
4.19. α-cholestanol, purity more than 99 % (purity must be checked by GC analysis). U.K.
4.20. α-cholestanol internal standard solution, 0,2 % solution (m/V) in ethyl acetate (point 4.11). U.K.
4.21. Phenolphthalein solution, 10 g/L in ethanol (point 4.10). U.K.
4.22. Carrier gas: hydrogen or helium, gas-chromatographic purity. U.K.
4.23. Auxiliary gases: hydrogen, helium, nitrogen and air, of gas-chromatographic purity. U.K.
4.24. n-Hexane (point 4.9)/ethyl ether (point 4.10) mixture 65:35 (V/V). U.K.
4.25. Silylation reagent, consisting of a 9:3:1 (V/V/V) mixture of pyridine/hexamethyl disilazane/trimethylchlorosilane.] U.K.