Trimyristin

Chemistry 214 Clark College

Trimyristin Extraction and Saponification Page 1 of 4

Trimyristin: A Fat from Nutmeg This lab was developed by Dr. Tom Goodwin at the Toad Suck Institute for Green Organic Chemistry at Hendrix College in Conway, Arkansas. (http://www.hendrix.edu/chemistry/green.htm)

REFERENCES 1. “Zero Effluent Laboratory”, L. R. Corwin, R. J. Roth, T. H. Morton, Brown University 2. “Soap from Nutmeg: An Integrated Introductory Organic Chemistry Laboratory Experiment”, M.

C. S. De Mattos, D. E. Nicodem, J. Chem. Educ. 2002, 79, 9495; 3. “Organic Chemistry”, 4th Ed., Brown, Foote and Iverson, Brooks/Cole Publishing, Belmont, CA,

2005.

INTRODUCTION Nutmeg is the common name of the seed from female Myristica fragrans, a tree native to the Spice Islands (the Moluccas, in the East Indies, now part of Indonesia). When the fruit of the tree matures, it splits in two, exposing a single seed sheathed in a crimson aril. The aril, a fibrous covering, constitutes the spice known as mace, and the seed is a whole nutmeg.

Nutmeg has been known as a spice in the Middle East for at least 2500 years, but it was not introduced into Europe until the Middle Ages. Dutch and Portuguese merchants monopolized the European nutmeg trade until 1843, when French and British interests succeeded in cultivating nutmeg trees in the West Indies. There is a decided difference between East Indian and West Indian nutmeg.

Like many other spices, nutmeg has a long history of medicinal use. Preparations of nutmeg have been used as analgesics, digestive stimulants, aphrodisiacs, amenorrheal agents, and hypnotics. Medicinal use has declined since the 18th century, save for a brief period at the end of the 18th century, when it was rumored to be an effective abortifacient. Several medical journals of this period reported a high incidence of nutmeg poisoning among women.

The nutmeg is about half cellulose. The other major components are fats, 25-40%, and essential oils, 8-15%. The former constitute the major portion of the expressed oil of nutmeg (obtained by distilling the seed with steam). Both of these components contain thousands of different compounds, only a handful of which have been identified. The expressed oil, also known as nutmeg butter, is composed principally of a lipid called trimyristin, the structural formula of which is illustrated in reaction 1. This fat also occurs in other plant and animal products, such as coconut oil and cow’s milk.

Trimyristin is an example of fundamental type of fat known as a triglyceride. Hydrolysis of one mole of a triglyceride affords one mole of 1,2,3-propanetriol (glycerin) and 3 moles of fatty acids, which are carboxylic acids containing the functional group at the end of a long alkyl chain. In addition to trimyristin (which is odorless when pure), solvent extraction of nutmeg yields as major components the three compounds illustrated below, which are responsible for most of the characteristic nutmeg odor. Myristicin occurs in much higher concentration than elemicin or safrole.

O

H3C(H2C)12 O O (CH2)12CH3

O

O

H3C(H2C)12 Otrimyristin

H3CO

H3CO

H3CO

OCH3

myristicin

elemicin safroleO

O

O

O



Myristicin has been studied pharmacologically. In mice it inhibits the action of monoamine oxidase, an important enzyme in the central nervous system, and a distinct psychotomimetic effect has been detected in man. The effect of myristicin is reported to be stronger when the other constituents of the volatile oil are present. It has been suggested that the other components do not themselves contribute to the psychotropic activity, but promote absorption of myristicin by irritating the gastrointestinal tract. One hypothesis regarding the hallucinogenic activity of allylbenzene derivatives is that they are metabolized to compounds related to amphetamine. As for many pharmacologically active substances, the difference between an effective dose and a toxic dose is not great. An after effect of ingestion of large quantities of nutmeg or myristicin is a lasting aversion to its taste or smell. PROCEDURE Extraction of Trimyristin:

1. Add 20 ml of petroleum ether to powdered nutmeg (approx.10 g, weighed to nearest 0.1 g) in a 50 ml round-bottomed flask. (Note: “petroleum ether” is an historical name, and a misnomer. This solvent is actually a mixture of low molecular weight alkanes, and not an ether at all. It has properties much like gasoline, so you should avoid flames, keep the solvent vapors away from hot surfaces, avoid inhalation, and avoid skin contact.)

2. Add a boiling stone, lightly grease the glass joint, and reflux the mixture GENTLY (keep the heat low) for 30 minutes (30 minutes of actual boiling time) under a water-cooled condenser. Record frequent observations in your notebook.

3. At the end of the reflux period, turn off the heat, and move the heating mantle away from the round-bottomed flask so that the flask can cool. Allow the flask to remain undisturbed while it cools, and the insolubles settle to the bottom.

4. When the flask is cool to the touch, pipet the liquid away from the solid. The liquid solution is what we want to keep, since it contains the extracted trimyristin. Put a small latex bulb on a short Pasteur pipet and, without disturbing the precipitate, transfer as much of the liquid as you can to a pre-weighed, clean, dry, 100 mL round-bottomed flask. NOTE: YOU MUST MINIMIZE THE TRANSFER DISTANCE FROM ONE ROUND-BOTTOMED FLASK TO ANOTHER, OR RISK LOSING SOLUTION AS IT SQUIRTS OR DRIPS FROM THE PIPET. Try to avoid getting solution on the ground glass joint of the flask.

5. Now to transfer the remaining liquid, your objective is to place the tip of the pipet on the inside bottom of the round-bottomed flask, without getting any of the brown solid into the pipet. This works best if, as you slowly plunge the tip through the liquid and solid, you slowly depress the bulb so that bubbles are slowly expelled through the tip to expel any solid that tries to enter. Once the pipet tip is seated on the flask bottom, gently expel any remaining air from the bulb, then slowly allow the bulb to inflate. If you do this carefully, the solid particles will be excluded from the pipet, and the liquid will slowly seep in. Once the latex bulb is fully inflated (i.e., no longer sucking up liquid), you need to transfer the liquid to the 100 mL flask. Repeat the transfer steps if necessary until all the solution is transferred. Dispose of the brown insolubles in the labeled waste container in the hood; shake or scrape out as much as you can. Do not put water into the disposal flask. Rinse flask with water and rinse down the sink. It will not hurt if some brown nutmeg leftovers go down the sink. Remember, people eat this stuff (it’s a green lab, with brown nutmeg!).



Removal of Pet Ether by Distillation

1. Wipe any residue off of the ground glass joint of the 100 mL distilling flask if necessary, then add a boiling stone. Distill the solvent using a simple distillation set-up. Collect the distilled solvent in a 25 mL round-bottomed flask. Petroleum ether has a very low boiling point, so very little heat is needed. Record boiling point in your lab notebook. You will need to watch carefully to judge when to stop the distillation. Remember that you started with approximately 25 mL of petroleum ether. At the end of your distillation, the 25 mL collection flask should be about 3/4 full. As soon as the liquid quits distilling and dripping into the collection flask, the heat should be turned off and removed. Allow the distillation flask to cool so that it is not warm to the touch. Dispose of the distilled petroleum ether in the waste container provided in one of the hoods.

Alternately, remove the petroleum ether by rotary evaporation.

2. Use a gentle stream of air to evaporate the last traces of petroleum ether from your solid residue. Reweigh the flask to determine your crude yield.

Purification by Recrystallization 1. Weigh 1 g of the crude, unpurified trimyristin into a clean, dry, large test tube. Add 4 mL of 95%

ethanol and a boiling stone, and then heat the mixture in a hot water bath. You may hold the tube with a test tube clamp as it heats in the water. You should stir with a small metal spatula to mix and to aid in dissolving.

2. Once the solid is completely dissolved, remove the test tube from the hot water, remove the boiling stone, and clamp the test tube or set it in a small beaker (to avoid tipping over) to allow the solution to cool to room temperature, or until you see white solid trimyristin begin to precipitate. Then, cool the test tube in ice to complete the precipitation.

3. Vacuum filter to separate the solid trimyristin.

4. Transfer your white, solid, odorless trimyristin carefully onto a pre-weighed watch glass and save it in your lab drawer. This product will be used for taking melting point and as the starting material for our next reaction, saponification (soap making) of the fat. [NOTE: Before taking the melting point, the recrystallized trimyristin should dry exposed to the air for at least overnight.] This is likely going to be the stopping point for the first week of lab.



Saponification (“Soap-making” by Alkaline Hydrolysis of an Ester) (Be sure to read the section on base-promoted ester hydrolysis in your text.)

O

H3C(H2C)12 O O (CH2)12CH3

O

O

H3C(H2C)12 O

trimyristin(a triglyceride)

NaOH, !

ethanol(! = heat)

ONa

O

3

+ HO OH

OH

sodium myristate

glycerin (glycerol)(1,2,3-propanetriol)

You will need to save around 25 mg of recrystallized trimyristin for a melting point determination. [NOTE: Before taking the melting point, the recrystallized trimyristin should dry exposed to the air for at least overnight, so you will need to wait until the next lab period to obtain the melting point.]

1. Weigh out your remaining recrystallized trimyristin for the saponification and transfer it to a clean 25 mL round-bottomed flask. You need 1.25 equiv of NaOH for the saponification; calculate the volume of 0.25 M NaOH needed to equal this after you get a mass of dried, recrystallized trimyristin. To the round-bottomed flask, add the calculated volume of a 0.25 M solution of NaOH in 95% ethanol. Add a boiling stone and gently reflux the mixture for 15 min (i.e., 15 min of actual gentle boiling). During the reflux period, a white solid precipitate should appear.

2. After cooling to room temperature, use ~5 mL of water to aid in the transfer of the mixture from the round-bottomed flask to a small clean beaker. Add 5 mL of saturated NaCl solution, mix well, and then vacuum-filter the solid soap with a Büchner funnel. Rinse the solid on the filter paper with 4 mL of cold water, then continue to draw air through it for a few minutes to dry the soap.

3. Determine the mass of your product.

Tests on Sodium Myristate 1. Dissolve ~200 mg of your sodium myristate in 20 mL of water. Transfer 5 mL of the soap

solution to each of 2 small test tubes. To one test tube, add a drop of vegetable oil, cork the tube, and shake to mix. Record your observations. To the other test tube, add several drops of a 1% aqueous solution of CaCl2 (“hard water”). Record your observations.

For your report

1. Tabulate your results. Include %fat content from the nutmeg and a percent yield for the saponification reaction

2. Write out the mechanism for the base-catalyzed saponification of trimyristin. Include all steps to go completely from trimyristin to 3 equivalents of sodium myristate and glycerin.

3. Write an experimental section for this experiment. Remember – each new compound should have its own paragraph, but only each new compound!

4. Discuss your observations from the tests on sodium myristate. What did you see? What do you think occurred in each case? How does the structure of sodium myristate account for this behavior? (Note – your answer to this question should be a decent paragraph…. A few short statements are not satisfactory. Structures and diagrams would help!)

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Trimyristin