2. Exhaustive Dewaxing by Soxhlet Extraction6
The equipment used for exhaustive dewaxing consists of a heat source (heating mantle and Variac controller), round bottom flask for a solvent reservoir, Soxhlet extractor, sintered-glass thimble or disposable extraction thimble, anti-bumping chips and a condenser (see Fig. 1). Note that the narrow siphon arm (parts 6 and 7 in Fig. 1) is very delicate and prone to breakage, requiring careful handling.
Put 0.5-1 g of tomato skin (obtained in step 1.) in a mortar, and grind the sample to a coarse powder with a pestle unless the samples are to be used for AFM measurements (Section 5). Fill a sintered-glass or disposable thimble approximately halfway with the sample and use tweezers to place it carefully at the base of the extraction column.
Attach the condenser and wrap it with aluminum foil. Heat the methanol solvent (ACS grade) in the presence of a few anti-bumping chips until it boils gently and refluxes on the walls of the flask. Cover the solvent reservoir with both glass wool and aluminum foil. Check the process for an hour, adjusting the Variac voltage so that the reservoir accumulates about one drop per second and siphoning occurs within the Soxhlet apparatus when the thimble is full.
Continue the extraction process for 12 h, then lower the heating mantle and allow the apparatus to cool down. Remove the extractor and reservoir as a single unit to dispose of the solvent. Use tweezers to raise the thimble to just below the neck of the extraction column, drain excess solvent from it, and place the thimble on a clean surface. Tilt the extraction column to allow siphoning into the flask below. Disconnect the flask and pour the waste into a labeled waste solvent receptacle.
Repeat steps 2.3 and 2.4 for successive solvents of progressively diminishing polarity, e.g., chloroform and hexane for 12 h in each case.
Allow the tomato cutin sample to dry inside the thimble, either by blowing a stream of nitrogen gas over it or by placing it in a vacuum oven at room temperature. Finally, measure the mass of the dry sample and store it at room temperature in a screw-top jar sealed with parafilm.