Division 6.3 Methylation of the sample

Cool all reagents and apparatus to between 0 and 4 ^oC for two hours. Into the external compartment of the diazomethane apparatus place 1,2 ml of the solution obtained in 6.2 and 0,1 ml of methanol (4.4). Place about 200 mg of diazald (4.2) in the central reservoir, add 1 ml of carbitol (4.5) and 1 ml of diethyl ether (4.3) and dissolve. Assemble the apparatus, half immerse the apparatus in a bath at 0 ^oC and introduce by syringe about 1 ml of cooled potassium hydroxide solution (4.7) into the central reservoir. Ensure that the yellow colour formed from the formation of diazomethane persists. If the yellow colour does not persist, repeat the methylation with a further 200 mg of diazald (4.2)^f1.

The apparatus is removed from the bath after 15 minutes then left closed at ambient temperature for 12 hours. Open the apparatus, react the excess diazomethane by adding a few drops of a 10 % (v/v) solution of formic acid in ethyl acetate (4.15) and transfer the organic solution to a 25 ml volumetric flask. Make up to volume with hexane (4.8).

Inject 1,5 μl of this solution into the chromatograph.