P3 - Trimyristin from nutmeg protocol PDF

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Trimyristin from nutmeg protocol...

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P3 – Isolation of Trimyristin from nutmeg (Adapted from F. Lugemwa, Molecules 2012, 17, 9274-9282)

You will use various extraction techniques to isolate a natural product (trimyristin) from nutmeg. You will characterise your product using IR spectroscopy and determine a melting point. Background Nutmeg is a spice derived from the seed of the nutmeg tree Myristica Fragrans and is commonly used in cooking. A reddish-brown solid, nutmeg is a complex mixture of naturally occurring organic compounds and its extracts are used in perfumes and as components in pharmaceuticals.

Figure 1: Chemical structure of trimyristin, a fragrant ester found in seeds of the nutmeg tree Myristica Fragrans.

One of the principal organic compounds responsible for the fragrance of nutmeg is trimyristin, an ester derived from the condensation of myristic acid and glycerol. This condensation reaction is shown below:

Figure 2: Condensation reaction of myristic acid and glycerol, forming trimyristin and water.

The separation or isolation of individual organic compounds from nutmeg is accomplished by a variety of techniques including steam distillation and extraction. In this experiment, we will use a specific type of extraction to remove the trimyristin from the nutmeg: solid‐liquid extraction.

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Extractions, either liquid‐liquid or solid‐liquid, take advantage of molecular solubility. A molecule’s structure and chemical properties such as polarity govern the types of solvents it will dissolve in. A good rule to remember is: ‘like dissolves like’. This means that polar substances will have better solubility in polar solvents while nonpolar solvents dissolve nonpolar molecules. Think about the solubility of trimyristin based on its chemical structure. Although the ester groups of trimyristin certainly contain polar bonds, the large alkane chains attached to the ester groups are not polar. Organic solvents like hot ether or dichloromethane followed by crystallization from acetone are traditionally used for trimyristin extraction. However, in this session you will use a more eco-friendly alternative, a mixture of ethyl acetate: ethanol: water (ratio 4.5: 4.5 : 1.0). The extraction is carried out under ‘reflux conditions’ (see figure). This setup is used when mixtures need to be heated for a long time at boiling point, as it prevents the loss of solvent due to evaporation. A condenser is attached to the boiling flask and is clamped in an upright position, the "reflux position", and cooling water is circulated to cause the vapours to condense as they rise up the condenser and thus prevent them from escaping. The upper level of the vapours in the condenser can often be seen as a reflux line or ring. The direction of flow of the water should be such that the condenser will fill with the cooling water; water should enter the condenser at the bottom and leave from the top. It is good practice to ensure that the heating source can easily be removed, either by raising the set up (using a laboratory jack) or ensure the assembly can be lifted out of the heating apparatus. Experimental Procedure

Access to fume cupboard needed for this experiment.

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Safety Wear lab coat, gloves and safety glasses at all times during this experiment.  

Use the fume cupboard for the reflux extraction and the hot filtration step. Ethyl acetate and ethanol are extremely flammable liquids:

A. Extraction of trimyristin 1. Set up a reflux apparatus in the fume cupboard; use a heating mantle for your heat source. 2. Weigh 2.0 gr of ground nutmeg and transfer this to a round bottomed flask. 3. Add three boiling stones (anti-bumping granules) and 25 mL of extraction solvent, a mixture of ethyl acetate, ethanol and water (4.5:4.5:1.0 ratio). 4. Ask your instructor to check the setup before you carry on with the extraction. Make sure the water flows through the condenser and check for leaks. 5. Heat to a gentle reflux (low boil; you should see a vapour ring in the bottom part of the condenser!) and then continue to reflux for at least 45 minutes. Don’t rush this step – it takes a while to unlock the trimyristin from the nutmeg! B. Filtration and crystallisation of trimyristin 6. While waiting for the reflux extraction, you can set up the vacuum filtration apparatus. It is important to have this ready when the reflux extraction is finished, as you need to filter the nutmeg solution while it is still hot. 7. Collect a small Buchner filter, a Buchner flask, and a vacuum pump. Assemble this according to the figure shown. 8. When finishing the reflux extraction, remove the round bottomed flask from the condenser (careful, hot!). Vacuum filter the mixture while still hot (in the fume cupboard). 9. Quickly transfer the filtrate to a 25 mL beaker and leave to stand at room temperature. 10. When the solution cools down, you should see the formation of crystals. The rate of cooling has a role in determining the size of 3

the crystals: fast cooling will tend to generate more crystals of relatively small dimensions, slow cooling allows larger crystals to form. Usually the best compromise of speed, convenience, and crystal quality, is to let the solution cool to room temperature on the bench first. 11. To ensure maximum recovery of crystals, the solution should be cooled in an ice–water bath after the solution has been allowed to cool to room temperature. (If needed, crystal formation can be encouraged by scratching the inner wall of the beaker with a glass stirring rod or spatula). 12. After crystallisation, vacuum filter the cold mixture and wash the crystals with a small amount (5 mL) of ice cold extraction solution (ethyl acetate: ethanol: water mixture). 13. Leave the air current passing for a while through the filter until it is dry. Weigh the amount of product obtained and determine the yield.

IR Spectroscopy: Run IR spectra of your isolated product and compare these with the literature. Your lab demonstrator will show you how to do this. Good link to general IR discussion website: http://www.science.oregonstate.edu/~gablek/CH335/Chapter10/IR.htm Melting point: Determine the melting point of your isolated product (see below for methodology). Take 3 measurements and report an average) and compare this with reference data from the literature. 1. Press the open end of the capillary tube onto your solid which should transfer some solid into the tube. Invert the tube and tap the sealed end gently onto a hard surface so the solid drops down to the bottom. Repeat this until you have a depth of 2 mm of sample in the sealed end of the tube. 2. You need to heat your sample relatively fast to a ‘plateau temperature’ of 40 °C. Above the plateau, the sample will continue to be heated but at a slower rate of 2-3 degrees per minute which will allow you to record an accurate melting range of your compound. 3. The first temperature is recorded when the first drop of liquid is apparent. The second temperature is recorded when the entire mass of crystals has been converted into a liquid.

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Record your first temperature here

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Record your second temperature here °C

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Give the melting range of your compound here °C

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4. Press stop to finish heating and allow the instrument to cool. Remove your sample tube and dispose in the glass waste bin. 5. Determine the melting point (range) of your isolated product (take 3 measurements and report an average) and compare this with reference data from the literature.

unsure.

Post-lab Questions: 1. What is the structural difference between saturated, mono-unsaturated, and polyunsaturated fats? Of which kind is trimyristin?

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2. How much trimyristin did you crystallize and what is the percent yield from nutmeg? Does this % agree with the literature? 3. What is the melting point of your final product? Compare this with the reported melting point from the literature. 4. Show the IR spectrum of your final product and discuss the major bands. 5. How pure do you think your isolated trimyristin is? Explain your answer. 6. Suggest how you can improve the percent yield of trimyristin.

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