Chemistry
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Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory
Chapters
Summary November 7th, 2018
Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.
Transcript
This technique represents an inherently practical and efficient strategy for natural products extraction in isolation chemistry. And we think it's a useful and complimentary extraction tool. Because this is a rapid flow process, the extract is only heated for a short period and the extraction of intractable plant pigments which often complicate purification is minimized.
We think that this technique is well suited to enabling chemo taxonomic sampling in addition to medicinal chemistry by facilitating bio prospecting which is the search for valuable molecules in nature. Visual demonstration of this method is important as a sample preparation in extraction steps are most effectively explained visually. This also reinforces the simplicity of the extraction and its appropriate use in the undergraduate laboratory environment.
Place 12.5 grams of coarsely ground cloves into a 250 milliliter beaker. Add 12.5 grams of sand and mix well. Next, collect a portafilter and load the basket with the entire clove and sand mixture.
Use a tamper to lightly compress the sample making sure not to compress it too much which would prevent fluid from flowing. Load the portafilter into the espresso machine and place a clear 250 milliliter beaker beneath it. Add a solution containing 30 percent ethanol in water to the water tank.
Using the espresso machine, collect 100 milliliters of the extract. Let the portafilter finish dripping and then remove it from the espresso machine. Use a spatula to remove the clove grinds from the portafilter and discard them into a solid waste container.
Rinse out any residual solids with tap water in the sink. Transfer the beaker containing the clove extracts to an ice bath and let it cool until the temperature reduced to at least 30 degrees celsius. In a fume hood, pour the cooled extract into a 250 milliliter separatory funnel.
Add 30 milliliters of hexane and mix by shaking gently. Place the separatory funnel in a ring clamp that is fitted to a retort stand and allow the aqueous and organic layers to separate. It can take up to 10 minutes for the layers to separate so we recommend that you carry out solvent optimization of TLCs while you wait.
Collect the aqueous layer back into the 250 milliliter beaker. Transfer the organic layer which contains the product to a clean 250 milliliter conical flask. Then pour the aqueous face pack into the separatory funnel.
Extract the aqueous layer two additional times with hexane. Using 30 milliliters of hexane each time. Combine the organic layers into the same 250 milliliter conical flask after each extraction.
After the third liquid extraction pour the combined organic extract into the separatory funnel. Wash the organic extract by adding 100 milliliters of water and shaking vigorously. Collect the organic layer into a clean 250 milliliter conical flask.
And then dry it by adding magnesium sulfate and swirling. Filter the dried mixture through fluted filter paper contained in a glass funnel into the round bottom flask. Using a rotary evaporator, evaporate the solvent from the collected filtrate.
Then weigh the flask containing the resultant oil. Add 10 milliliters of hexane to the round bottom flask containing the crude eugenol containing extract. Pour the resulting solution into a 250 milliliter separatory funnel.
Rinse the round bottom flask with 10 milliliters of hexane and add this to the separatory funnel. Extract the hexane solution twice with three molar aqueous sodium hydroxide via liquid liquid extraction. Using 25 milliliters of sodium hydroxide for each extraction.
Collect the aqueous layers from each extraction and combine them in a 250 milliliter conical flask. Collect the organic layer in a 50 milliliter conical flask. Then dry it by adding magnesium sulfate to the flask and swirling it.
Filter the solution through fluted filter paper contained in a glass funnel into a pre-weighed 100 milliliter round bottom flask. Discard the solid residue then use a rotary evaporator to remove the solvent. After this weigh the round bottom flask which now contains the resulting oil on a top pan balance.
Next place the conical flask that contains the combined aqueous layers into an ice water bath. Swirl the flask while slowly adding a 10 molar solution of hydrochloric acid until a white emulsion is formed. Use a pipette to transfer a drop of this solution onto Congo red paper to test its acidity.
The paper should turn blue. After this transfer the milky aqueous emulsion to a 250 milliliter separating flask. Make sure that the emulsion is at room temperature and then perform liquid liquid extraction twice using 30 milliliters of hexane per extraction.
Combine the two hexane extracts into a clean 100 milliliter conical flask. Add magnesium sulfate to dry the solution. Filter the solution through fluted filter paper contained in a glass funnel into the pre-weighed 100 milliliter round bottom flask.
Then use a rotary evaporator to remove the solvent. After this weigh the round bottom flask which now contains the resulting oil on a top pan balance. Using an electric spice grinder grind 10 grams of correa reflexa leaves for 20 to 30 seconds.
Transfer the ground plant material to a 250 milliliter beaker. Add approximately two grams of coarse sand. Mix the sand and plant material together and then transfer it into the basket of a portafilter.
Use a tamper to lightly compress the sample making sure not to compress it too much. Add about 300 milliliters of 35 percent ethanol in water to the espresso machine tank. Load the portafilter into the espresso machine and place a 250 milliliter beaker beneath it.
Then collect approximately 100 milliliters of extract. Wait for about one minute then collect another 100 milliliters of extract. Transfer this mixture to an ice bath to cool.
Use a rotary evaporator with a water bath temperature of 40 degrees celsius to evaporate the ethanol. Next transfer the aqueous extract to a seperatory funnel. Extract the solution twice with ethyl acetate using 50 milliliters of ethyl acetate per extraction.
Combine the organic extracts. Add magnesium sulfate and swirl the flask to dry the solution. Then use a centered glass funnel to filter the solution and use a rotary evaporator to obtain the crude extract.
In this procedure students are allocated a TLC solvent ratio of acetone and cyclohexane and provided with pure standards of eugenol and acetyl-eugenol. TLC analysis is performed and the effects of solvent composition on the retention factor are considered. The optimum solvent compositions are seen to typically range between five and 20 percent acetone cyclohexane.
With a delta retention factor of 0.1 to 0.2. The different acid based properties of the two major organic molecules are then exploited to separate them by liquid liquid extraction. Typically eugenol was isolated in a yield of 45 to 65 percent of the crude extract while acetyl-eugenol was isolated in a yield of five to 10 percent.
Students then utilized the optimized eluant to determine the success of their liquid liquid extraction by comparison of their extracts to the pure reference samples by TLC. Advanced students then subject half their isolated crude oil to flash column chromatography. While the complete separation of eugenol from acetyl-eugenol is rarely achieved due to their close retention factors, a few fractions containing pure eugenol are generally collected.
Following this procedure, methods like nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry can be performed characterized the isolated compounds. Don't forget, working with hot liquids, relatively high pressures and corrosive liquids can be hazardous so precautions such as wearing personal protective equipment should be taken.
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