Background: The nutmeg seed is unusual in that over 75% of its total lipid content is made up of a triglyceride which contains a single fatty acid residue. By careful extraction and crystallization, it is possible to obtain this triglyceride in pure form. Then, base saponification of the triglyceride hydrolyzes the ester bonds and releases the sodium salt of the fatty acids. These molecules are “soaps”. Neutralization of the soaps with acid generates free fatty acids, which can be crystallized and identified by melting point determination. The recovered fatty acid sample can also be tested for unsaturation using a test for oxidation by potassium permanganate.
Objectives: after this experiment, you will be able to:
- Extract the lipid fraction from a natural source
- Saponify a triglyceride and isolate the soaps
- Neutralize the soaps to prepare free fatty acids
Determine the physical (melting point) and chemical (unsaturation test) characteristics of the recovered fatty acids
Solvents: diethyl ether (peroxide free), acetone, 95% ethanol, methanol
14 M NaOH
KMnO4 unsaturation test solution (0.5 g KMnO4 dissolved in 100. mL 0.1 M H2SO4) Stearic and oleic acid solutions (0.5 g free acid dissolved in 100. mL reagent-grade acetone)
CAUTION: Ether is volatile, flammable and an inhalation hazard (an anesthetic). DO NOT leave ether-containing vessels open to the air; close with a cork or stopper except during transfer. Work in a hood to evaporate ether.
Instructions: You may work in pairs or a group of no more than 3 for this experiment.
Part A: Isolation of the triglyceride from nutmeg
Weigh out about 5 g of ground nutmeg and place it in a 50 mL Erlenmeyer flask. Add 20 mL of ether, stir well and allow the mixture to stand for 5 minutes. Filter by gravity into a 100 mL beaker. Return the solid material to the flask and extract with another 20 mL portion of ether. Filter and repeat the extraction with a third 20 mL portion of ether, combining the extracts.
Evaporate the combined extracts in a hot water bath on a hot plate IN THE HOOD until essentially all the ether is gone. Add 50 mL of acetone and warm slightly to completely dissolve the material. Add 0.05 g decolorizing carbon, heat the mixture just to the boiling point and filter rapidly while hot into a 125 mL Erlenmeyer flask.
Allow the flask and its contents to cool to room temperature and then chill it in an ice bath for at least 15 minutes to get maximum crystallization. Filter by suction to gather the crystals, allow them to air dry then determine the mass and melting point of the product.
Part B: hydrolysis of the triglyceride and characterization of the fatty acid component
Place about 1 g of pure triglyceride in a 125 mL Erlenmeyer flask. Add 2.0 mL of 14 M NaOH and then 15 mL of 95% ethanol. Mix by swirling. Then, insert a glass conical funnel in the neck of the flask and heat in a hot water bath on a hot plate for 30 – 40 minutes. Remove the funnel and continue heating until essentially all of the ethanol has evaporated.
Add 50 mL of deionized water and heat the mixture to dissolve all of the soap. Then add to this solution enough 4 M HCl to neutralize the NaOH used for the saponification. Check to make certain the solution is acidic. Chill the mixture in an ice bath and allow it to stand until crystallization of the fatty acid is complete.
Filter by suction to gather the solid, transfer it to a 50 mL Erlenmeyer flask and add 25 mL of methanol. Heat to between 45 and 50°C then add deionized water drop-wise until the mixture is faintly cloudy. Set the mixture aside to slowly cool and crystallize. After about 30 minutes, stopper the flask and place it in a refrigerator for at least 30 minutes more.
Prepare a small funnel for suction filtration. Filter the cold solution off of the solid. Air dry the solid then determine the mass and melting point of the recovered fatty acid. Note: you are particularly interested in the range over which the solid melts since this is an indication of purity.
Test the fatty acid for unsaturation as follows: Place about 10 mg of solid in a small test tube and dissolve it in about 2 mL of reagent-grade acetone. Add 1 drop of potassium permanganate solution and shake gently. If the purple color fades within 30 seconds, repeat the permanganate addition drop- wise until the solution remains purple. Record the number of drops needed for the solution to remain colored. Perform similar tests on 2 “standards” – 0.5% solutions of stearic acid and oleic acid in acetone.
Determine the % recovery of triglyceride. Determine the % recovery of fatty acid from the triglyceride used in the saponification. Based on the melting point determinations and your observations during melting, were the triglyceride and the fatty acid samples pure? Explain. Interpret the test results. Based on your data, what is the probable identity of the fatty acid recovered from the hydrolyzed nutmeg triglyceride? Justify your identification.
- What is the major triglyceride found in nutmeg? Draw this triglyceride. Provide a citation.
- Write a balanced equation for the reaction of the above triglyceride with NaOH. Provide a citation.
- Write a balanced equation for the reaction of oleic acid and steric acid with potassium permanganate. Explain the difference in this test for the two standards.
- What other triglycerides can be found in nutmeg? Provide a citation.
- What other types or classes of compounds might have been present in the nutmeg extract? These compounds could have been extracted into organic solvent from the NaOH solution after saponification. These components of the lipid fraction are sometimes referred to as “nonsaponifiable lipids”. Provide a citation.
- Would you expect the free fatty acid obtained from nutmeg to be completely saturated or completely unsaturated?
- Triglycerides release fatty acyl salts upon saponification. A second compound is also released from the triglyceride. Name it and draw its structural formula. How was this second triglyceride component separated from the free fatty acid?
- Write a complete structural formula for the “soap” obtained when the nutmeg triglyceride was saponified.
Prepare the table(s) for the results to be obtained in this experiment as part of your pre-lab.