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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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><tbody><tr><td>espresso machines</td><td>Breville/Sunbeam</td><td>Breville espresso machine model 800ES / Sunbeam EM3820 Caf&amp;eacute; Espresso II</td><td></td></tr><tr><td>rotary evporators</td><td>Buchi and Heidolph</td><td></td><td></td></tr><tr><td>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><td>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><td>sand</td><td>Ajax</td><td>1199</td><td></td></tr><tr><td>ethanol</td><td>Redoc Chemicals</td><td>E95 F3</td><td></td></tr><tr><td>hexanes</td><td>Ajax</td><td>251</td><td></td></tr><tr><td>magnesium sulfate</td><td>Ajax</td><td>1548</td><td></td></tr><tr><td>diethyl ether</td><td>Merck</td><td>1009215000</td><td></td></tr><tr><td>silica on aluminium TLC plates</td><td>Merck</td><td>1055540001</td><td></td></tr><tr><td>eugenol</td><td>Merck</td><td>1069620100</td><td></td></tr><tr><td>eugenyl acetate</td><td>Aldrich</td><td>W246905</td><td></td></tr><tr><td>acetone</td><td>Redox Chemicals</td><td>Aceton13</td><td></td></tr><tr><td>cyclohexane</td><td>ChemSupply</td><td>CA019</td><td></td></tr><tr><td>silica gel 60</td><td>Trajan</td><td>5134312</td><td>40 - 63um (230-400mesh)</td></tr><tr><td>Congo red paper</td><td>ChemSupply</td><td>IS070-100S</td><td></td></tr><tr><td>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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Chemistry

14.3K 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

Basic Organic Chemistry Techniques

Glassware in Organic Chemistry
There are standard pieces of glassware that are used in the organic chemistry lab. Beakers and Erlenmeyer flasks are typically used for simple mixing or to hold solvents but they should not be used to measure volume, unless only an approximate volume is needed. Glassware can be divided into the following categories:
<ol>
	<li style="font-size:17px">Volumetric Glassware &#8211; This includes glassware such as graduated cylinders, volumetric glass pipettes, and volumetric flasks. The purpose of these items is to measure or dispense a precise volume of a substance. A graduated cylinder is generally used to measure volumes in an organic lab. The graduated cylinder has a long cylindrical shape with volume markings on the side. It is essential to use the smallest graduated cylinder possible for the volume needed in order to minimize error. For example, a 25-mL graduated cylinder is adequate to measure 20 mL of reagent, but it is not accurate to measure a smaller volume, like 5 mL. However, do not use a 25-mL graduated cylinder to measure 100 mL by measuring four 25-mL volumes; it is better to use the 100-mL graduated cylinder.</li>
	<li style="font-size:17px">Reaction Containment &#8211; These specialized pieces can be used to house an organic chemical reaction. Round-bottom flasks are an example of this glassware. Unlike Erlenmeyer flasks, round-bottom flasks provide more surface area and therefore provide more uniform heating or cooling. Round-bottom flasks are used to perform reactions under high heat or vacuum because their round shape is more resistant to cracking. In this category are also specialized glassware known as condensers, which are used to collect gaseous vapors and condense them back into the liquid phase to be collected.</li>
	<li style="font-size:17px">Filtration &#8211; These pieces of glassware are mainly used to help isolate precipitates out of a reaction mixture. To isolate solid precipitates, a filtration apparatus is required. The simplest setup utilizes a glass funnel attached to an Erlenmeyer flask. Inside the funnel is a piece of filter paper. The reaction mixture containing the solid is poured into the funnel, and gravity will pull the liquid down into the flask, leaving behind the solid. This is known as gravity filtration. However, this setup may require a long period of time to filter the solid. A vacuum filtration setup replaces the Erlenmeyer flask with a vacuum flask with a side arm and the glass funnel with a porcelain filter funnel known as a B&#252;chner funnel. This side arm is attached to a vacuum source. Like before, the reaction mixture is poured into the filter funnel and the filtration is aided by force from the vacuum.</li>
	<li style="font-size:17px">Separation &#8211; These specialized flasks or funnels are used mainly for liquid-liquid phase extractions, which is a technique that allows for the separation of products by using solvents with different polarities or hydrophobicities. These immiscible liquids will form a heterogeneous solution that separates into two phases. Separatory funnels have a valve known as a stopcock at the bottom, allowing for separation of the phases by pouring them into a new container.</li>
	<li style="font-size:17px">Transport &#8211; This glassware mostly consists of bottles or vials with a corresponding cap. This glassware is used primarily to transport potentially hazardous material safely.</li>
	<li style="font-size:17px">General use &#8211; This category is comprised of glassware such as beakers, Erlenmeyer flasks, glass rods, and any other miscellaneous pieces.</li>
</ol>
Weighing Organic Reagents
Organic reagents can come in multiple forms, and it is important to practice good lab technique when obtaining these reagents for use in experiments. Always review a reagent&#39;s MSDS to determine potential hazards when working with it. Solid reagents should be measured out using weighing boats or weighing paper and the appropriate type of balance.
If a precise measurement is required, use an analytical balance. If a large amount of reagent is to be used, use a top-loading balance. Place a weighing boat or weighing paper on the balance and push the &#8220;tare&#8221; button. The tare feature will null the mass of the weighing paper. Always use spatulas to transfer solid reagents from the supply to the weighing boat. Never reuse the same spatula for different reagents as this can contaminate the stock reagents. And never return excess reagent to the stock bottle. Instead, discard it in the appropriate way as instructed.
When transferring the solid to the appropriate vessel, use a small funnel to carefully pour the solid inside. If any solid remains, use a small amount of the solvent to be used on the weighing boat and transfer it into the flask.
Organic liquids can be measured by using volumetric glassware, such as a graduated cylinder. Calculate the volume of the liquid using its density, then tare the reaction flask on the balance. Use a pipet or graduated cylinder to transfer the volume directly to the reaction flask. Follow the guidelines for the specific type of glassware. Never reuse the same piece of glassware for multiple reagents and never return excess reagent to the liquid stock bottles. For liquids, c
Heating Organic Reactions
Sometimes heat is required to enable an organic chemical reaction to occur. In the general lab setting, heat is commonly applied using a Bunsen burner with a direct gas flame. In organic chemistry labs, an open flame from a Bunsen burner can create a dangerous situation. Organic reagents, particularly solvents, are highly combustible and some form vapors with relative ease. For that reason, Bunsen burners are not used in organic chemistry labs.
Instead, heating baths, hotplates, or mantles are used to provide an indirect source of heat. Hotplates with magnetic stirring functionality are used to heat beakers and Erlenmeyer flasks. Heating mantles are designed to safely heat a round-bottom flask with varying volumes. Water baths are used when the temperature of the reaction does not need to exceed 100 &#176;C. A reaction in glassware is immersed in a water bath that is heated by a hot plate. The temperature is modulated to the appropriate range. If the required temperature needs to exceed 100 &#176;C but not 250 &#176;C, a silicone mantle can be used. If the temperature must exceed 250 &#176;C, a sand bath can be used.
Many reactions require being heated to a certain temperature for long periods of time in order for it to proceed. However, if a reaction is heated for a long period of time, the solvent may evaporate, causing a loss of reaction solution. Instead, a reflux setup is often used, which uses a round-bottom flask containing a solvent. The boiling point of the solvent overlaps with the optimal temperature of the reaction. The round-bottom flask is clamped to a stand, and a condenser is fitted onto the flask. Cold water flows through the condenser from the bottom arm to the top arm while the mixture is heated and stirred. As the mixture is heated, the solvent evaporates and then condenses back into the flask, preserving the reaction volume.

Podstawowe techniki chemii organicznej

Glassware in Organic Chemistry
There are standard pieces of glassware that are used in the organic chemistry lab. Beakers and Erlenmeyer flasks are ...

Education

JoVE Lab Manual

Steam Distillation

<h4>Steam Distillation </h4>
Steam distillation is a separation technique that harnesses the low boiling point property of immiscible mixtures. It is predominately used to separate temperature-sensitive organic molecules from a non-volatile contaminant. The organic molecule must be immiscible in water.
In steam distillation, the immiscible mixture is heated to boiling, causing the distillation of both water and the volatile organic compounds. This means that the gaseous mixture travels upwards to a condenser, which then condenses the vapor to liquid so that it can be collected. In contrast to simple distillation, steam distillation uses a water reservoir to replenish water in the heated mixture throughout the process. The immiscible organic component is slowly distilled along with the water, while the non-volatile component remains in the heated mixture. Once the organic component is distilled, it can then be separated from the water using liquid-liquid extraction.
<h4>Vapor Pressure of a Mixture</h4>
For a miscible mixture that forms a homogeneous solution, the vapor pressure of each component is dependent on the vapor pressure of the pure component and its mole fraction in the liquid mixture according to Raoult&#8217;s law.
pA = pA*xA
where pA is the vapor pressure of one liquid component in a miscible liquid mixture, pA* is the vapor pressure of the pure liquid, and xA is the mole fraction of that liquid in the mixture, which is equal to nA/nt. nA is the number of moles of the individual liquid in the mixture, and nt is the total number of moles of all the liquids in the mixture.
The total vapor pressure above the miscible liquid mixture is equal to the sum of the partial vapor pressure of each component in it, which is known as Dalton&#8217;s law. The vapor pressure of a liquid increases with temperature as more molecules gain kinetic energy to escape the liquid phase to the gas phase. In a miscible mixture containing two liquids, the total pressure can be described as:
P = pA + pB
where pA and pB are the vapor pressures of liquid A liquid B, respectively, above the mixture. P is the total vapor pressure of both liquids above the mixture. Combining the equations describes the relationship between the total vapor pressure of the solution and the mole fraction of the individual components:
P = pA*xA + pB*xB
In an immiscible mixture, where the components form a heterogeneous mixture, the vapor pressures of each component contribute independently to the total vapor pressure. Thus, the total vapor pressure is equal to the sum of the individual pure vapor pressures. In an immiscible mixture composed of two liquids, the total pressure is defined as the vapor pressure of the first liquid plus the vapor pressure of the second liquid.
P = pA* + pB*
<h4>Boiling Point of an Immiscible Mixture</h4>
As a liquid is heated, the vapor pressure increases. Each component in a mixture has its own boiling point. In a mixture of miscible liquids, boiling occurs at a temperature between the boiling points of the constituent liquids.
For an immiscible mixture, boiling occurs at a much lower temperature than the boiling points of the individual components. As each individual component contributes independently, less heat is required to increase the total vapor pressure to the atmospheric pressure.
For example, consider the immiscible mixture of benzene and water. The boiling point of benzene at normal atmospheric pressure is 80.1 &#176;C, and the boiling point of water at normal atmospheric pressure is 100 &#176;C. The solution boils when the total vapor pressure reaches 760 mm Hg (normal atmospheric pressure). At 69.3 &#176;C, the vapor pressure of water is 227 mm Hg and the benzene vapor pressure is 533 mm Hg, which in total equals the necessary 760 mm Hg required to boil. This is well below the boiling point of either individual component.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silderberg, M.S. (2009). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill.</li>
</ol>

Steam Distillation: Extraction of Essential Oil from Orange Peel - Concept

Destylacja z parą wodną

Steam Distillation 
Steam distillation is a separation technique that harnesses the low boiling point property of immiscible mixtures. It is predominately ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biochemistry

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the deep ocean. Roles of minerals are critical in many hydrothermal organic geochemical processes. Traditional hydrothermal methodology, which includes using reactors made of gold, titanium, platinum, or stainless-steel, is usually associated with the high cost or undesired metal catalytic effects. Recently, there is a growing tendency for using the cost-effective and inert quartz or fused silica glass tubes in hydrothermal experiments. Here, we provide a protocol for carrying out organic-mineral hydrothermal experiments in silica tubes, and we describe the essential steps in the sample preparation, experimental setup, products separation, and quantitative analysis. We also demonstrate an experiment using a model organic compound, nitrobenzene, to show the effect of an iron-containing mineral, magnetite, on its degradation under a specific hydrothermal condition. This technique can be applied to study complex organic-mineral hydrothermal interactions in a relatively simple laboratory system.

Earth-abundant minerals play important roles in the natural hydrothermal systems. Here, we describe a reliable and cost-effective method for the experimental investigation of organic-mineral interactions under hydrothermal conditions.

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

Environment

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

Solid-Liquid Extraction

Source: Laboratory of Dr. Jay Deiner &#8212; City University of New York
Extraction is a crucial step in most chemical analyses. It entails removing the analyte from its sample matrix and passing it into the phase required for spectroscopic or chromatographic identification and quantification. When the sample is a solid and the required phase for analysis is a liquid, the process is called solid-liquid extraction. A simple and broadly applicable form of solid-liquid extraction entails combining the solid with a solvent in which the analyte is soluble. Through agitation, the analyte partitions into the liquid phase, which may then be separated from the solid through filtration. The choice of solvent must be made based on the solubility of the target analyte, and on the balance of cost, safety, and environmental concerns.

Solid-Liquid Extraction: Principle, Procedure, Applications

Source: Laboratory of Dr. Jay Deiner &#8212; City University of New York
Extraction is a crucial step in most chemical analyses. It entails removing the ...

JoVE Science Education

Organic Chemistry

Extraction of Biomarkers from Sediments - Accelerated Solvent Extraction

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The distribution of a group of organic biomarkers called glycerol-dialkyl glycerol-tetraethers (GDGTs), produced by a suite of archaea and bacteria, were found in modern sediments to change in a predictable manner in response to air or water temperature1,2. Therefore, the distribution of these biomarkers in a sequence of sediments of known age can be used to reconstruct the evolution of air and/or water temperature on decadal to millennial timescales (Figure 1). The production of long high-resolution records of past climates, called paleoclimatology, depends on the rapid analysis of hundreds, possibly thousands of samples. Older extraction techniques, such as sonication or Soxhlet, are too slow. However, the newer Accelerated Solvent Extraction technique was designed with efficiency in mind.
<img alt="Figure 1" src="/files/ftp_upload/10097/10097fig1.jpg" /> 
Figure 1. An example of a paleoclimate record showing changes in sea surface temperature (SST) in the eastern Mediterranean Sea during the past ~27,000 years3. This record comprises ~115 samples and is based on the isoprenoidal GDGT-based TEX86 SST proxy.

Accelerated Solvent Extraction of Total Lipid from Sediments

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The distribution of a group of organic biomarkers called glycerol-dialkyl ...

Environmental Sciences

Earth Science

Boiling Points

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Put on the necessary personal protective equipment, including a lab coat, chemical splash goggles, and gloves.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">The students will need to use both acetone and ethanol in their experiment. Label a glass bottle with the solvent name, and then pour a smaller volume of solvent from the stock bottle into the glass bottle, ensuring that there is enough for each group to have about 1 mL. Do the same for the other solvent and then place these bottles in a communal hood.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Lab stand</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Lab jack</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Rubber band</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Medium 3-prong clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3-4&#160;&#160;&#160;&#160;Glass capillary tubes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Pipette bulb</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;1-mL volumetric pipettes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;Glass test tubes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;250-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;25-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Digital thermometer</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
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</ol>

Determination of Boiling Points by Capillary Method - Prep

Temperatury wrzenia

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of the LaboratoryExpand
	Here, we show the laboratory preparation ...

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

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