The authors acknowledge the School of Natural Sciences - Chemistry, University of Tasmania and the School of Chemistry, The University of Sydney for financial support. B.J.D. and J.J. thank the Australian Government for Research Training Program Scholarships.

NOTE: It is advisable that all procedures are performed in a fume hood. Students must wear appropriate personal protective equipment at all times in the laboratory and the safety data sheets (SDS) associated with each reagent must be consulted before use.
1. PHWE of cloves: isolation of eugenol and acetyleugenol
<ol>
	<li>Extraction of eugenol and acetyleugenol from cloves
	<ol>
		<li>Place coarsely ground cloves (12.5 g) in a 250-mL beaker.</li>
		<li>Add sand (12.5 g) to the clove grinds and mix well.</li>
		<li>Collect a portafilter (sample compartment) and load the basket with the entire clove-sand mixture. Lightly compress the sample with the tamper. 
		NOTE: Do not compress the mixture too much or fluid will not flow through.</li>
		<li>Position the portafilter into the espresso machine and place a clean 250-mL beaker beneath it. Add a 30% ethanol/H2O solution to the water tank of the espresso machine if it is less than half full.</li>
		<li>Use the espresso machine to collect 100 mL of the extract. 
		NOTE: Consult an instructor if the machine appears to be clogged.</li>
		<li>Allow the portafilter to finish dripping and then remove it from the espresso machine. 
		CAUTION: The grinds and surrounding metal areas will be hot.</li>
		<li>Using a spatula, remove the clove grinds from the portafilter and discard into the waste bin.</li>
		<li>Rinse out the residual solids from the portafilter with H2O under a tap in the sink and return it for the next person to use.</li>
		<li>Cool the clove extract in an ice bath until the temperature has reduced to at least 30 &#176;C.</li>
		<li>Place the extract into a 250-mL separatory funnel, add 30 mL of hexane and shake gently.</li>
		<li>Place the separating funnel in a ring clamp fitted to a retort stand and allow the aqueous and organic layers to separate then collect the aqueous (lower) layer back into the 250-mL beaker. 
		NOTE: It can take to 10 minutes for the layers to separate. Students are advised to carry out solvent optimization of TLCs while waiting for the first separation to occur (see steps 1.2).</li>
		<li>Transfer the organic (top) layer (which contains the product) to a clean 250-mL conical flask, and then pour the bottom (aqueous) layer back into the separatory funnel.</li>
		<li>Extract the aqueous layer a further two times with hexane (2x 30 mL).</li>
		<li>Combine the organic (top) layers into the same flask after each extraction.</li>
		<li>After the third liquid-liquid extraction, pour the combined organic extract into the separatory funnel, and wash with 100 mL of H2O by shaking vigorously. Collect the organic (top) layer into a clean 250-mL conical flask and dry by adding MgSO4 and swirling the flask.</li>
		<li>Filter the ensuing mixture through fluted filter paper contained in a glass funnel into a pre-weighed 250-mL round-bottom flask. 
		NOTE: The solid residue (hydrated MgSO4) can be discarded in the waste.</li>
		<li>Evaporate the solvent (hexane) from the collected filtrate using a rotary evaporator (water bath temperature: 60 &#176;C, vacuum pressure: 350 mbar) and re-weigh the flask containing the resultant oil.</li>
	</ol>
	</li>
	<li>Optimization of thin-layer chromatography (TLC) solvent system 
	NOTE: As a group, students will be assigned a solvent system from 100:0 acetone:cyclohexane to 0:100 acetone:cyclohexane by the instructor&#160;to identify the ratio that provides the maximum resolution of eugenol from acetyleugenol.&#160;
	<ol>
		<li>Obtain a TLC reference solution of pure eugenol and acetyleugenol. 
		NOTE: The necessary TLC reference solutions of pure eugenol and acetyleugenol were prepared by lab technicians prior to the lab session.</li>
		<li>On a TLC plate, mark a baseline ~1.5 cm from the bottom with a soft pencil. Mark off three equally spaced points.</li>
		<li>Use a TLC spotter to spot one drop of pure eugenol TLC reference solution in one lane, one spot of pure acetyleugenol TLC reference solution in the third lane and a spot of each in the second lane (the co-spot).</li>
		<li>Check for the presence of eugenol and acetyleugenol on the TLC plate by viewing the plate under a UV lamp (254 nm) in the TLC viewing cabinet. 
		NOTE: There should be small (1-2 mm wide) black spots on the plate where the TLC reference solutions were spotted. If there are no spots or the spots appear faint, apply another spot of the appropriate TLC solution until a black spot is observed under UV light.</li>
		<li>Add 10 mL of the allocated solvent mixture to a clean, dry TLC jar. 
		NOTE: Ensure the solvent height in the jar does not exceed ~1 cm.</li>
		<li>Using tweezers, place the prepared TLC plate into the TLC jar. Close the lid of the jar. 
		NOTE: The solvent must lie below the baseline of the TLC plate.</li>
		<li>Allow the solvent to travel up the TLC plate. Once the solvent is ~1 cm from the top of the plate, remove the TLC plate from the jar with tweezers and mark the line of the solvent front with a pencil.</li>
		<li>Allow the solvent to evaporate from the TLC plate (~1 min) then view the TLC plate under a UV lamp (254 nm). Using a pencil, circle the black spots observed on the TLC plate.</li>
		<li>Calculate the retention factor (Rf) of eugenol and acetyleugenol by dividing the distance travelled by the compound by the distance travelled by the solvent.</li>
		<li>Calculate the difference between the Rf values for eugenol and acetyleugenol (&#916;Rf).</li>
		<li>Share the results with the rest of the class. Record the retention values acquired by other students with other solvent ratios.</li>
		<li>Identify which solvent system will be best to analyze the crude eugenol solution and subsequent purification steps. 
		NOTE: The best TLC solvent ratio will provide the greatest separation between eugenol and acetyleugenol denoted by the largest &#916;Rf value. If &#916;Rf is plotted against solvent composition, the graph should resemble a bell curve.</li>
	</ol>
	</li>
	<li>Separation of eugenol and acetyleugenol by liquid-liquid extraction
	<ol>
		<li>Add hexane (10 mL) to the crude eugenol-containing extract obtained from step 1.1.17, and pour the ensuing solution into a 250-mL separatory funnel.</li>
		<li>Rinse the round-bottom flask with hexane (10 mL) and add this to the separatory funnel.</li>
		<li>Extract the hexane solution with 3 M aqueous NaOH (2 x&#160;25 mL) via liquid-liquid extraction. Collect and combine the aqueous bottom layers in a 250-mL conical flask from each extraction. Collect the organic layer in a 50-mL conical flask and dry by adding MgSO4 and swirling the flask. 
		CAUTION: NaOH is corrosive. Avoid any contact with skin. 
		NOTE: Acetyleugenol remains in the organic layer, while eugenol is now in the alkaline aqueous extract (bottom layers).</li>
		<li>Retain the organic layer (organic solution A) for later TLC analysis.</li>
		<li>Swirl the conical flask that contains the alkaline aqueous fraction from step 1.3.3 in an ice-water bath and slowly add 10 M aqueous HCl until a white emulsion is formed; check its acidity with Congo red paper, using a pipette to transfer a drop of the solution onto the pH paper (it should turn blue). 
		CAUTION: HCl is corrosive. Avoid any contact with skin. Addition of HCl can cause vigorous bubbling, HCl should be added carefully, keeping the conical flask on ice. 
		NOTE: A total of 20-30 mL of HCl (10 M of an aqueous solution) will be required.</li>
		<li>Extract the milky aqueous emulsion with hexane (2 x 30 mL), using liquid-liquid extraction in a 250 mL separating flask. Make sure that the temperature of the aqueous extract is at room temperature or below before adding the hexane. Combine the two hexane extracts into a clean 100 mL conical flask. 
		NOTE: The eugenol will now be in the combined organic (top) layers (organic solution B).</li>
		<li>Add MgSO4 to dry organic solution B.</li>
		<li>Analyze organic solution A, organic solution B, pure eugenol TLC reference and acetyleugenol TLC reference by TLC using the optimized TLC solvent ratio identified in the previous session.</li>
		<li>Filter organic solution A and B through fluted filter paper into separate&#160;pre-weighed 250-mL round-bottom flasks. Discard the solid residue (hydrated MgSO4) in the waste.</li>
		<li>Remove the solvent from the round-bottom flasks using a rotary evaporator (water bath temperature: 60 &#176;C, vacuum pressure: 350 mbar).</li>
		<li>Add diethyl ether (5 mL) to each&#160;round-bottom flask and transfer the purified acetyleugenol (organic solution A)&#160; and eugenol (organic solution B) into an unlabeled, preweighed vial using a funnel.</li>
		<li>Rinse the flask with further diethyl ether (5 mL) into the vial. Evaporate the solvent using a rotary evaporator (water bath temperature: 50 &#176;C, vacuum pressure 800 mbar) with a vial attachment. Record the yield and label the vial appropriately.&#160;</li>
		<li>Analyze organic solution A, organic solution B, pure eugenol TLC reference and acetyleugenol TLC reference by TLC using the optimized TLC solvent ratio identified in the previous session. 
		NOTE: Aqueous solutions can be poured down the sink for disposal. Hexane and ether waste should be disposed of in the non-chlorinated organic waste bottles.</li>
	</ol>
	</li>
</ol>
2. PHWE of Correa reflexa : isolation of seselin and (+)-epoxysuberosin
<ol>
	<li>Session 1. PHWE of Correa reflexa
	<ol>
		<li>Grind Correa reflexa leaves (10 g) in an electric spice grinder and then transfer the ground plant material to a 250-mL beaker. 
		NOTE: Grinding should take 20-30 seconds.</li>
		<li>Add ~ 2 g of coarse sand to the beaker containing plant material.</li>
		<li>Mix and pack into the basket of the portafilter (sample compartment). Compress the sample with the tamper. 
		NOTE: Do not pack the sample too tightly.</li>
		<li>Add ~300 mL of 35% ethanol/H2O solution to the espresso machine tank.</li>
		<li>Position the portafilter into the espresso machine and place a clean 250-mL beaker beneath it.</li>
		<li>Collect ~100 mL of extract, wait for ~1 minute and then collect a further 100 mL. 
		CAUTION: The machine and extracts will be hot at this point.</li>
		<li>Cool this mixture in ice bath and evaporate the ethanol using a rotary evaporator (water bath temperature: ~40 &#176;C).</li>
		<li>Transfer the aqueous extract to a separatory funnel and extract with ethyl acetate (4x 50 mL). 
		NOTE: Time may be needed to allow emulsions to separate between extractions.</li>
		<li>Combine the organic extracts, dry by adding MgSO4 and swirling the flask, filter using a sintered glass funnel, and evaporate using a rotary evaporator (water bath temperature: ~35 &#176;C) to provide the crude extract.</li>
		<li>Obtain an 1H nuclear magnetic resonance (NMR) spectrum (see the instructor for assistance).11</li>
		<li>Perform TLC analysis of the crude extract to determine an appropriate solvent system to isolate the compounds that have been extracted. 
		NOTE: TLC analysis is performed by analogy to procedures outlined in step 1.2.</li>
	</ol>
	</li>
	<li>Session 2. Separation of seselin and (+)-epoxysuberosin by flash column chromatography11,19 
	NOTE: The following protocol involves the use of flash column chromatography for the separation of organic compounds. Please consult an instructor to demonstrate how to pack a flash silica gel column.
	<ol>
		<li>Place the column (~30 mm in diameter) in a clamp fitted to a retort stand. Place a 100-mL conical flask underneath the column.</li>
		<li>Fill the column with silica gel (60 &#956;m flash grade) to a level of ~10 cm and then add hexanes (~100 mL) to the column.</li>
		<li>Place a glass stopper in the column, remove the column from the clamp and shake to obtain a slurry. Place the column in the clamp and then allow the mixture to settle.</li>
		<li>Open the tap of the column and, using a gas adaptor attached to a compressed air line, empty the column to leave ~2 mm of solvent above the bed of silica gel. Remove the gas adaptor then close the tap.</li>
		<li>Use the hexanes collected in the conical flask (~5 mL) to wash down any silica gel from the walls of the column with a Pasteur pipette fitted with a rubber septum.</li>
		<li>Repeat step 2.2.4 then add a small layer of sand (~1 cm) to the column.</li>
		<li>Add dichloromethane (~ 1mL) to the flask containing the crude extract from step 2.1.9. Carefully load the ensuing solution onto the column using a Pasteur pipette fitted with a rubber septum. Open the tap of the column and allow the sample to adsorb onto the silica gel.</li>
		<li>Repeat step 2.2.7 a further two times.</li>
		<li>Carefully add hexanes (~20 mL) to the column. Repeat step 2.2.4.</li>
		<li>Carefully add (~180 mL of a 15% ethyl acetate/hexanes solution). Open the tap of the column and, using a gas adaptor attached to a compressed air line, empty the column to leave ~2 mm of solvent above the bed of silica gel, collecting the fractions into 10-mL test tubes. 
		NOTE: This will allow seselin to be isolated.</li>
		<li>Combine the test tube fractions containing seselin in a 250-mL round bottom flask and evaporate using a rotary evaporator (water bath temperature: ~35 &#176;C). 
		NOTE: TLC analysis is used to determine this and is performed by analogy to procedures outlined in step 1.2.</li>
		<li>Carefully add (~75 mL of a 25% ethyl acetate/hexanes solution). Open the tap of the column and, using a gas adaptor attached to a compressed air line, empty the column to leave ~2 mm of solvent above the bed of silica gel, collecting the fractions into 10-mL test tubes. 
		NOTE: This will allow (+)-epoxysuberosin to be isolated.</li>
		<li>Combine the test tube fractions containing (+)-epoxysuberosin in a 250-mL round bottom flask and evaporate using a rotary evaporator (water bath temperature: ~35 &#176;C). 
		NOTE: TLC analysis is used to determine this and is performed by analogy to procedures outlined in step 1.2.</li>
		<li>Samples of the isolated compounds are analyzed using NMR spectroscopy.11 
		NOTE: NMR spectroscopy experiments are performed by a lab technician.</li>
	</ol>
	</li>
</ol>

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J., Kilah, N. L., Jordan, G. J., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbutin+Derivatives+Isolated+from+Ancient+Proteaceae:+Potential+Phytochemical+Markers+Present+in+Bellendena,+Cenarrhenes+and+Persoonia+Genera.">Arbutin Derivatives Isolated from Ancient Proteaceae: Potential Phytochemical Markers Present in Bellendena, Cenarrhenes and Persoonia Genera.</a> Journal of Natural Products. 81, 1241-1251 (2018).</li><li>Deans, B. J., Tedone, L., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemical+profile+of+the+rare,+ancient+clone+Lomatia+tasmanica+and+comparison+to+other+endemic+Tasmanian+species+L.+tinctoria+and+L.+polymorpha.">Phytochemical profile of the rare, ancient clone Lomatia tasmanica and comparison to other endemic Tasmanian species L. tinctoria and L. polymorpha.</a> Phytochemistry. 153, 74-78 (2018).</li><li>Deans, B. J., Skierka, B., Karagiannakis, B. W., Vuong, D., Lacey, E., Smith, J. A., Bissember, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Siliquapyranone:+a+Tannic+Acid+Tetrahydropyran-2-one+Isolated+from+the+Leaves+of+Carob+(Ceratonia+siliqua)+by+Pressurised+Hot+Water+Extraction.">Siliquapyranone: a Tannic Acid Tetrahydropyran-2-one Isolated from the Leaves of Carob (Ceratonia siliqua) by Pressurised Hot Water Extraction.</a> Australian Journal of Chemistry. 71, (2018).</li><li>Olivier, W. J., Kilah, N. L., Horne, J., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ent-Labdane+Diterpenoids+from+Dodonaea+viscosa.">ent-Labdane Diterpenoids from Dodonaea viscosa.</a> Journal of Natural Products. 79, 3117-3126 (2016).</li><li>Rihak, K. J., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polygodial:+A+viable+natural+product+scaffold+for+the+rapid+synthesis+of+novel+polycyclic+pyrrole+and+pyrrolidine+derivatives.">Polygodial: A viable natural product scaffold for the rapid synthesis of novel polycyclic pyrrole and pyrrolidine derivatives.</a> Tetrahedron. 74, 1167-1174 (2018).</li><li>Ntamila, M. S., Hassanali, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Oil+of+Clove+and+Separation+of+Eugenol+and+Acetyl+Eugenol.">Isolation of Oil of Clove and Separation of Eugenol and Acetyl Eugenol.</a> Journal of Chemical Education. 53, 263 (1976).</li><li>Still, W. 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The authors have nothing to disclose.

The classical procedure for isolating eugenol from cloves by steam distillation has been part of the intermediate chemistry laboratory program at the University of Sydney for decades but was modernized to employ PHWE methodology in 2016 (Figure 1).9,18 This provided a number benefits. Firstly, utilizing household espresso machines in the laboratory environment immediately fascinated and engaged students by illustrating the applicatio...

The isolation and identification of natural products are of fundamental importance to the scientific community and society more generally.1 Bioprospecting, the search for valuable organic molecules found in nature,remains an indispensable process in the discovery of new drug leads and potential therapeutic agents. It is estimated that, from 1981-2014, ~75% of all approved small molecule pharmaceutical drugs were natural products, natural product-derived or natural product-inspired.1 Furthermore, natural products possess enormous structural and chemical diversity. For this reason, they also represent valuable chemical scaffolds that can be directly used in organic synthesis or in the development of chiral ligands and catalysts.2,3
Traditionally, relatively time-intensive procedures such as maceration, Soxhlet extraction, and steam distillation have been the mainstay of research focused on the isolation of secondary metabolites from plants.4 More modern extraction techniques, including accelerated solvent extraction, have focused on reducing extraction times and establishing greener protocols.4,5 In 2015, an original pressurized hot water extraction (PHWE) method was reported.6 This technique employed an unmodified household espresso machine to facilitate the rapid and particularly efficient extraction of shikimic acid from star anise. Espresso machines have been specifically designed and engineered to extract organic molecules from appropriately ground coffee beans. To achieve this, these instruments heat water at temperatures up to 96 &#176;C and at pressures of typically 9 bar.7 With this in mind, it is perhaps not surprising that espresso machines can be utilized to efficiently extract natural products from a range of plant material.
Subsequent studies involving a variety of terrestrial plant species have demonstrated the capacity of this PHWE technique to efficiently extract natural products across a relatively broad polarity range.6,8,9,10,11,12,13,14,15 Furthermore, compounds containing somewhat sensitive functional groups, such as aldehydes, epoxides, glycosides, and potentially epimerizable stereogenic centers were typically unaffected by the extraction process. The complementary nature of this technique relative to more traditional methods has also been demonstrated.12,16 This PHWE method has also been employed to isolate multi-gram quantities of natural products, which have been used to prepare novel natural product derivatives and in complex molecule synthesis more generally.8,11,17
It was identified that this new PHWE method could serve as a useful teaching tool that could be incorporated in the undergraduate laboratory. This primarily derives from the rapid nature of this technique relative to the traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. Consequently, this technique supplanted the traditional undergraduate chemistry laboratory experiment focused on the extraction of eugenol from cloves employing steam-distillation at the University of Tasmania.9,18 Since that time, variations of this experiment have been adopted by other universities and a modified experiment focusing on the PHWE of cloves now features in the undergraduate chemistry laboratory program at the University of Sydney (vide infra).
In order to demonstrate the practicality and feasibility of employing this new PHWE approach for teaching purposes, two protocols are presented as part of this study. The first part of this report highlights an experiment on the PHWE of eugenol and acetyleugenol from cloves which is part of the second-year undergraduate laboratory program at the University of Sydney (Figure 1). This experiment serves to introduce students to natural products chemistry while developing fundamental practical skills. The second part features an experiment on the PHWE of the endemic Australian plant species Correa reflexa which is part of the third-year undergraduate laboratory program at the University of Tasmania (Figure 2). This experiment is designed to simulate natural products bioprospecting and reinforce core laboratory techniques.11

<table border="0" cellpadding="0" cellspacing="0" style="width:1069px;" width="1070">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">espresso machines</td>
			<td style="width:167px;">Breville/Sunbeam</td>
			<td style="width:520px;">Breville espresso machine model 800ES / Sunbeam EM3820 Caf&#233; Espresso II</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">rotary evporators</td>
			<td style="width:167px;">Buchi and Heidolph</td>
			<td style="width:520px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cloves (plant material)</td>
			<td>Dijon Food Pty Ltd</td>
			<td></td>
			<td>Cloves must be ground in a food processor for students.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Correa reflexa (plant material)</td>
			<td>sample obtained in Tasmania</td>
			<td>Sample collected from mature shrubs in the Thomas Crawford Reserve at the University of Tasmania</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">sand</td>
			<td style="width:167px;">Ajax</td>
			<td style="width:520px;">1199</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ethanol</td>
			<td>Redoc Chemicals</td>
			<td>E95 F3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">hexanes</td>
			<td>Ajax</td>
			<td>251</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">magnesium sulfate</td>
			<td>Ajax</td>
			<td>1548</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">diethyl ether</td>
			<td>Merck</td>
			<td>1009215000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">silica on aluminium TLC plates</td>
			<td>Merck</td>
			<td>1055540001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">eugenol</td>
			<td>Merck</td>
			<td>1069620100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">eugenyl acetate</td>
			<td>Aldrich</td>
			<td>W246905</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">acetone</td>
			<td>Redox Chemicals</td>
			<td>Aceton13</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cyclohexane</td>
			<td>ChemSupply</td>
			<td>CA019</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">silica gel 60</td>
			<td>Trajan</td>
			<td>5134312</td>
			<td style="width:167px;">40 - 63um (230-400mesh)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Congo red paper</td>
			<td>ChemSupply</td>
			<td>IS070-100S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">32% hydrochloric acid</td>
			<td>Ajax</td>
			<td>256</td>
			<td></td>
		</tr>
	</tbody>
</table>

employing pressurized hot water extraction (phwe) to explore natural products chemistry in the undergraduate laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

PHWE of cloves. When attempting to perform the liquid-liquid extraction step, students often encountered emulsions (the addition of brine was typically not effective). At this stage, students were instructed to allow the mixture to stand in the separating funnel while they explored the effects of eluent composition on the separation of eugenol and acetyleugenol by TLC. It should be noted that hexane can be substituted with either heptane or dichloromethane in the liquid-liquid extraction ...

Watch this Scientific Journal Video about Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory at JoVE.com

Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

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.

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products ...

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Research

JoVE Journal

Chemistry

14.2K Views.  University of Tasmania. 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.

Video: Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, &#916;H) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.
In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine kcat and KM of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (&#916;Hint) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.

Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean urease.

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important structural element found in many complex natural products and pharmaceuticals. Owing to the continual discovery of new guanidine-containing natural products and designed small molecules, rapid and efficient guanidinylation methods are of keen interest to synthetic and medicinal organic chemists. Because the nucleophilicity and basicity of guanidines can affect subsequent chemical transformations, traditional, indirect guanidinylation is typically pursued. Indirect methods commonly employ multiple protection steps involving a latent amine precursor, such as an azide, phthalimide, or carbamate. By circumventing these circuitous methods and employing a direct guanidinylation reaction early in the synthetic sequence, it was possible to forge the linear terminal guanidine containing backbone of clavatadine A to realize a short and streamlined synthesis of this potent factor XIa inhibitor. In practice, guanidine hydrochloride is elaborated with a carefully constructed protecting array that is optimized to survive the synthetic steps to come. In the preparation of clavatadine A, direct guanidinylation of a commercially available diamine eliminated two unnecessary steps from its synthesis. Coupled with the wide variety of known guanidine protecting groups, direct guanidinylation evinces a succinct and efficient practicality inherent to methods that find a home in a synthetic chemist&#39;s toolbox.

Here, we present a protocol for direct, early stage guanidinylation that enables rapid total synthesis of aminoguanidine-containing small organic molecules. An advanced synthetic intermediate used in the synthesis of a blood coagulation factor XIa inhibitor was prepared using this protocol.

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Laboratory Production of Biofuels and Biochemicals from a Rapeseed Oil through Catalytic Cracking Conversion

The work is based on a reported study which investigates the processability of canola oil (bio-feed) in the presence of bitumen-derived heavy gas oil (HGO) for production of transportation fuels through a fluid catalytic cracking (FCC) route. Cracking experiments are performed with a fully automated reaction unit at a fixed weight hourly space velocity (WHSV) of 8 hr-1, 490-530 &#176;C, and catalyst/oil ratios of 4-12 g/g. When a feed is in contact with catalyst in the fluid-bed reactor, cracking takes place generating gaseous, liquid, and solid products. The vapor produced is condensed and collected in a liquid receiver at -15 &#176;C. The non-condensable effluent is first directed to a vessel and is sent, after homogenization, to an on-line gas chromatograph (GC) for refinery gas analysis. The coke deposited on the catalyst is determined in situ by burning the spent catalyst in air at high temperatures. Levels of CO2 are measured quantitatively via an infrared (IR) cell, and are converted to coke yield. Liquid samples in the receivers are analyzed by GC for simulated distillation to determine the amounts in different boiling ranges, i.e., IBP-221 &#176;C (gasoline), 221-343 &#176;C (light cycle oil), and 343 &#176;C+ (heavy cycle oil). Cracking of a feed containing canola oil generates water, which appears at the bottom of a liquid receiver and on its inner wall. Recovery of water on the wall is achieved through washing with methanol followed by Karl Fischer titration for water content. Basic results reported include conversion (the portion of the feed converted to gas and liquid product with a boiling point below 221 &#176;C, coke, and water, if present) and yields of dry gas (H2-C2's, CO, and CO2), liquefied petroleum gas (C3-C4), gasoline, light cycle oil, heavy cycle oil, coke, and water, if present.

This paper presents an experimental method to produce biofuels and biochemicals from canola oil mixed with a fossil-based feed in the presence of a catalyst at mild temperatures. Gaseous, liquid, and solid products from a reaction unit are quantified and characterized. Conversion and individual product yields are calculated and reported.

The work is based on a reported study which investigates the processability of canola oil (bio-feed) in the presence of bitumen-derived heavy gas oil ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface (SALVI) and Scanning Electron Microscopy (SEM). This paper describes the method and key steps in integrating the vacuum compatible SAVLI to SEM and obtaining secondary electron (SE) images of particles in liquid in high vacuum. Energy dispersive x-ray spectroscopy (EDX) is used to obtain elemental analysis of particles in liquid and control samples including deionized (DI) water only and an empty channel as well. Synthesized boehmite (AlOOH) particles suspended in liquid are used as a model in the liquid SEM illustration. The results demonstrate that the particles can be imaged in the SE mode with good resolution (i.e., 400 nm). The AlOOH EDX spectrum shows significant signal from the aluminum (Al) when compared with the DI water and the empty channel control. In situ liquid SEM is a powerful technique to study particles in liquid with many exciting applications. This procedure aims to provide technical know-how in order to conduct liquid SEM imaging and EDX analysis using SALVI and to reduce potential pitfalls when using this approach.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface ...

Minimum Burning Pressures of Water-based Emulsion Explosives

This manuscript describes a protocol to measure the minimum pressure required for sustained burning of water-based emulsion explosives. Pumping water-based emulsion explosives for blasting applications can be very hazardous, as demonstrated by a number of pump accidents around the globe in the last decades, including some that resulted in fatalities. In Canada, the recognition of this hazard has led to the development of pumping guidelines that were endorsed by both the explosives industry and the Explosive Regulatory Division of the Canadian government. In these guidelines, it was noted that the minimum burning pressures (MBP) measured in a laboratory would provide a good guide to characterize the behaviour of these products in pumping systems. The same guidelines also call for the design of pump systems that prevent, whenever possible, pressures from exceeding the MBP of the product being pumped. At the time of publication of these guidelines, a methodology existed for measuring such MBP values but it had never been validated to measure the MBP of ammonium nitrate water-based emulsions (AWEs). AWEs are now used much more widely than any other water-based explosives and precursors in on-site bulk loading operations.
The Canadian Explosives Research Laboratory (CanmetCERL) has been conducting research over the last ten years to develop a validated testing protocol to measure and interpret representative MBP values for AWEs. The test, as it is performed today, will be described and the critical components will be justified by reference to recent published data. Results of MBP measurements, for a range of AWE products, will be presented. Inclusion of the MBP test in the test standards for the authorization of high explosives in Canada will also be discussed.

We present an apparatus based on hot-wire ignition in a pressurized enclosure and an associated methodology to measure the minimum pressure required to induce sustained combustion in water-based emulsion explosives. This method improves product characterization to allow one to use them more safely during pumping and mixing operations.