Objective Statement: The objective of this lab is to use steam distillation to extract natural products from plant materials. The products of the plant materials will be vaporized out of the material through high-pressure and high-temperature steam for collection. And then use dichloromethane in an extraction process to analyze the product by mass and through thin-layer chromatography.

Compounds of Study:

Eugenol

Formal Weight: 164.204 g/mol

Boiling Point: 254 Degrees Celsius

Pre-Lab Questions:

1. High-end perfumes are very expensive because the natural products used to create the scent are very expensive. Even though steam distillation is an effective way to isolate such products, what do you think the price of such products means about the efficiency of the isolation?

That the efficiency of the isolation requires a lot of time, effort, and material for a high and pure yield.

2. Most of the desired products are terpenoid in character. They belong to a class of compounds known as the terpenes. Conduct an image search of terpenes and describe in good detail what structural components all terpenes have in common.

Terpenes have linking isoprene structures in chain or ring forms.

3. In a paragraph (4-5 sentences), describe how the process of steam distillation works.

The process of steam distillation works by separating a water-immiscible oil of low volatility from a non-volatile contaminant. By using water vapor through steam, our low-volatility oil gets extracted out of the solution without being boiled out. These will leave the non-volatile contaminants in a heated solution, not boiled, so they stay in a separate flask and do not proceed through the steam distillation apparatus. Doing so allows for the extractions of the oil to condense and essentially be carried out of the solution to be condensated and there forever removed in a new solution.

4. The amount of a natural product that is “carried” by steam depends upon the molecular math of the product. Solve the following scenario using the equations presented in the text. A mixture of ethyl iodide (CH3CH2I, bp 72.3 °C) and water boils at 63.7 °C. What weight of ethyl iodide would be carried over by 1 g of steam during steam distillation? You will need to use the Vapor Pressure of the water table that is copied on the next page of this document. What the equations in the text represent is that the mixture will boil when the contribution of both components adds to 760 torr. You will find the partial pressure contribution from water using the table. Put the numbers you know into the equation and set the number you don’t know to “X” then solve the equation for X. The calculation requires you to use mathematical principles learned in algebra. Take a picture of your calculation and upload it into labarchives. You must show all your work.

Answer below.

5. Sometimes the molecular weight of the isolated natural product is unknown. It is important to know the molecular weight so that the product can be identified. Using the same equations, one can solve for this mass. Consider the following scenario - the condensate from a steam distillation contains 8 g of an unknown compound and 18 g of water. The mixture steam was distilled at 98 °C. What is the molecular weight of the unknown? Use the vapor pressure of the water table and the equation in your book to solve. You will solve by the same method you used to solve for question #3 you just have a different unknown value. Take a picture of your calculation and upload it into labarchives. You must show all your work.

Answer below.

6. Extraction is described as partitioning. This is exactly how we describe what happens during TLC – the material partitions between the mobile phase and the continuous phase. In extraction, the partitioning is between two liquid phases.

A. Considering that your two solvents are water and dichloromethane, if the natural product material partitions into the dichloromethane, what does that mean about the polarity of the compound? Briefly explain.

This would mean that the polarity of the compound will be the same as dichloromethane. Dichloromethane is a polar compound, so our natural product material is also polar.

B. Will the partition coefficient/distribution coefficient be greater than 1 or smaller than 1? Briefly explain.

The partition coefficient measures how hydrophilic (water-loving) or how hydrophobic (water-fearing) a chemical substance is. And if the natural product does not partition with water, then it is hydrophobic and therefore has a partition coefficient greater than 1. This can be checked as the concentration of our natural product will be more concentrated in our dichloromethane, than in water.

7. You will have two layers within your separatory funnel, what do you need to know about the two phases to determine which is forming the top layer and which is forming the bottom layer?

The solvent with a higher density will be on the bottom and the lower density will be on the top.

8. What are the seven steps for using the separatory funnel? Use the steps for extraction above and create your 7-step process for the separatory funnel portion of the extraction process.

Place distillate into a large separatory funnel.
Add 1/3 volume of distillate in dichloromethane to a separatory funnel.
Put the cover on the funnel and invert the funnel around 20 times to mix the distillate and dichloromethane, allowing pressure to escape through the stopcock of the funnel every 4-5 inversions.
Let the mixture settle. Remove emulsions through a saturated salt solution if necessary.
Remove the stopper and drain the two layers into separate Erlenmeyer flasks.
Determine the identity of the layers by adding a few drops of dichloromethane to each. The layer that dissolves the dichloromethane is the organic layer, and the other is the polar aqueous layer.
Add magnesium sulfate to the dichloromethane layer until the solid no longer "clumps" but "swirls". The magnesium sulfate binds to the water, thus leaving the solvent anhydrous and easier to evaporate.

9. One only holds a separatory funnel in their hands during mixing. What is the proper way for the separatory funnel to be secured at other times?

To be firmly held on a ring stand, and with a beaker below to catch solutions that leak or spill.

10. Although dichloromethane is not soluble in water, there can be dipole-dipole interaction between dichloromethane and water because there are polar bonds. A small amount of water therefore is going to dissolve into the dichloromethane. Magnesium or sodium sulfate is added because it is a solid hygroscopic material. What is a hygroscopic material and therefore what is its purpose in the extraction process?

A hygroscopic material is a substance that can adsorb and absorb water from the surrounding environment. Its purpose in the extraction process is to remove the small amounts of water that dissolved into the dichloromethane layer to purify the organic solution.

Procedures:

Activity #1:

Set up steam distillation apparatus, greasing all joints to ensure a tight fit so no steam would escape. (see picture below)
20g of cloves were placed into a 1000mL flask filled about halfway with water and placed onto a heating mantle.
A steam flask was filled with about 500mL of water on a separate hot plate.
Heating mantle set to just under 50 percent and the hot plate put at max heat.
After 45 minutes of the steam flask boiling, no distillation occurred so switched out the heating mantle. The heating mantle was not producing heat.
Covered steam distillation set up in tin foil. To keep heat insulated
After 1 hour and 30 minutes the distillation flask was boiling. The water turned yellow to a maroon color as steam distillation occurred in the distillation flask.
At around 1 hour and 45 minutes first drops started to come out.
Collected 125 mL of distillate. Slightly opaque in color and had an extremely strong clove scent.
Turned off the heat and immediately disconnected the steam generation system from the distillate system.

Activity #2

Placed the distillate into a large separatory funnel.
Added 40 mL dichloromethane into the separatory funnel.
Put the cover on the funnel and invert the funnel around 20 times to mix the distillate and dichloromethane, and allow pressure to escape through the stopcock of the funnel every 4-5 inversions.
Let the mixture settle. No emulsions formed.
Removed the stopper and drained the two layers into separate Erlenmeyer flasks.
Determined the identity of the layers by adding a few drops of dichloromethane to each layer. The layer that dissolved in the dichloromethane is the organic layer, and the other is the polar aqueous layer.
Added magnesium sulfate to the organic layer until the solid no longer clumped together. The magnesium sulfate binds to the water, thus leaving the solvent anhydrous and easier to evaporate, which also makes the solution clear.
Filtered the magnesium sulfate from the organic layer by vacuum filtration into a Buchner flask.
Under vacuum pressure, evaporate the organic solvent using low heat. Keep heat low to ensure that the product is. The solution started to turn slightly turquoise.
See results below for mass in grams.

Activity #3

Grabbed an 8cm long piece of chromatography paper.
Placed a small dot of the concentrated clove oil about 1.5cm from the bottom of the chromatography paper.
Used a 3:5:1 hexane/ethyl acetate/solvent mixture in a liquid chromatography chamber.
Let the paper sit in the chamber until the solvent was around 1.5 cm from the top.
Results were analyzed with UV light. To ensure proper spotting

Results:

Initial amount of cloves: 20g
Distilled 125 mL of concentrated clove oil.
Mass of concentrated clove oil extracted after evaporation: 1.26g
Percent yield: (2.26g recovered /20g in flask) x 100 = 6.3%
Rf value: 0.324

Conclusions:

Steam distillation is a technique used to extract things like essential oils from plant compounds, by using a concept of volatile compounds. Votalie compounds like oils, can be vaporized at temperatures much lower than their boiling points through the use of evaporation from steam. Through my distillation processes of 20g of clove leaves, I collected 125 mL of distillate. After evaporation of that distillate, 2.26 g of clove oil was obtained. Using these values, a yield percentage of 6.3% was the result of my experiment. This percent yield is very low and is mainly because of two mishaps during my lavatory experiment. First, the heating mantle used to heat my distillation flask was broken and I did not realize this until a lot of the lab time had passed. This is the main reason why I only collected 125 mL of distillate and not 150 mL as the laboratory handout suggested. One of the joints connecting the distillation flask to the water condenser also became loose during the experiment, letting some of the volatile steam holding the essential clove oil. However, the use of thin-layer chromatography(TLC) did show that the 11.3g of concentrated clove oil distilled was pure. With analysis of UV lighting, only one spot was depicted on the TLC paper, and an Rf value of .324 was calculated. Putting the Rf value to the understanding that our solvent solution is also very nonpolar, due to the higher concentration of ethyl acetate, the isolated terpenes is a non-polar organic compound. This means that there was only one compound in the distillate solution after evaporation, or in other words, a pure compound.

Reflections:

One of the main lessons I learned from this lab was the understanding of equipment checks before and during laboratory experiments. Easily avoidable mistakes that cost me yield percentage could have been prevented or understood much earlier if I had paid attention/looked to see why I was not getting distillate past the expected time frame. Equipment checks are something I will be looking out for in my future labs to make sure I create a smooth and effective experiment.