Name: procedure for the transfer of polymer films onto porous substrates with minimized defects
Uploaded: 2019-06-22T14:00:00.000+00:00
Duration: 5 min 2 s
Description: Watch this Scientific Journal Video about Procedure for the Transfer of Polymer Films Onto Porous Substrates with Minimized Defects at JoVE.com

<p class="jove_content">This work was supported as part of the Advanced Materials for Energy-Water Systems (AMEWS) Center, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences. We gratefully acknowledge helpful discussions with Mark Stoykovich and Paul Nealey.</p>

<p class="jove_title">1. Fabrication of the transfer tool and drain chamber system</p>
<ol>
	<li>Attached (<strong>Supplementary Files 1</strong>, <strong>2</strong>) is the engineering drawing for the drain chamber assembly consisting of two parts: top and bottom. Model this device according to the specifications of the desired system (e.g., the outer diameter of the receiving substrate) and export as an STL file for 3D printing.</li>
	<li>For the top part, utilize a filament printer of choice and print in the lowest resolution possible, including scaffolding wherever necessary. Adhere to the recommended parameters of the printer. It is also recommended that the top part be printed using poly(lactic acid) (PLA) to minimize material shedding.</li>
	<li>For the bottom part, use an inkjet resin printer or filament printer with a build height as fine as 20 &#181;m.<br />
	NOTE: PLA is a suitable material which minimizes material shedding.</li>
	<li>Scrub and clean both parts with deionized water, ensuring the removal of any potential shedding material from the printing process. Sonication in deionized water is also recommended. Test the threading on the two parts to ensure a good fit.</li>
	<li>Complete the drain chamber with a size 117 neoprene O-ring and tubing of the parameters specified in the supporting documents (<strong>Supplementary Files 1</strong>, <strong>2</strong>). A schematic of the entire drain chamber assembly is shown in <strong class="xfig">Figure 1</strong>.</li>
	<li>Print the transfer tool using any filament printer at medium to fine resolution. There are two parts: clamp and loading arm.<br />
	NOTE: It is <strong>highly recommended</strong> that the transfer tool be printed using poly(lactic acid) (PLA), as other plastics can be poorly wetted and cause the wafer to become wet unexpectedly.</li>
	<li>Complete the clamp with a size 10 screw and then attach the clamp onto a laboratory jack.</li>
</ol>
<p class="jove_title">2. Initial mechanized deposition and membrane lift-off from donor substrate</p>
<ol>
	<li>Place a bare 25 mm-diameter AAO disc (or any arbitrary porous receiver substrate of choice) onto the bottom part of the drain chamber. Then, place the neoprene O-ring on top of the AAO disc and screw on the top part of the drain chamber.</li>
	<li>Rinse and/or sonicate the setup various times with deionized (DI) water. This helps to remove any dust and/or any remaining particulates from 3D printing.</li>
	<li>Place the piece of Si wafer with the transferable polymer stack (donor wafer) onto the lip of the transfer tool loading arm.</li>
	<li>Fill the drain chamber with 25 mL of DI water.</li>
	<li>Lower the laboratory jack so that the tool is dipped slowly into the entrance ramp of the drain chamber and that the donor silicon substrate is slowly submerged. Ensure that the wafer is submerged sufficiently for the membrane to completely delaminate and lift-off from the underlying donor substrate.<br />
	NOTE: Using a piece of Si wafer with no dust contamination will ensure easy separation from the donor substrate.</li>
	<li>Slowly raise the transfer tool out of the water and move it out of the way, making sure not to disturb the floating membrane.</li>
	<li>Coax the membrane into the opening of the chamber with tweezers. Placing the tweezer in water in front of membrane will guide it due to surface tension. Touching the floating membrane itself is not necessary and should be avoided.</li>
</ol>
<p class="jove_title">3. Meniscus-guided transfer to receiver substrate with the drain chamber system</p>
<ol>
	<li>Connect tubing to the outlet of the bottom part of the drain chamber. Attach this tubing to a 20 mL Luer-lock syringe.</li>
	<li>Obtain a syringe pump with withdrawing functionality. Place the syringe onto the pump and withdraw water at a rate of 1-2.5 mL/min until all the water has been drained out.</li>
	<li>After 10 min, the water should be completely removed from the drain chamber. If there is still any residual water within the chamber, reconnect the syringe and tubing and continue to withdraw any residual water.</li>
	<li>After complete drainage of the water, the membrane will now be placed at the center of the receiver substrate. Disconnect the drain chamber from the syringe pump and disassemble the drain chamber to remove the receiver substrate containing the membrane.<br />
	NOTE: The total process including set-up takes ~15 min. Reducing the working volume of water and increasing the drain rate can shorten this process.</li>
	<li>Allow the sample to dry completely at room temperature before further use in any application.</li>
</ol>

<ol>
	<li>Shah, A., Torres, P., Tscharner, R., Wyrsch, N., Keppner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photovoltaic+technology:+the+case+for+thin-film+solar+cells.">Photovoltaic technology: the case for thin-film solar cells.</a> <em style='font-style: italic'>Science</em>. <b>285</b> (5428), 692-698 (1999).</li><li>Kim, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-colour+quantum+dot+displays+fabricated+by+transfer+printing.">Full-colour quantum dot displays fabricated by transfer printing.</a> <em style='font-style: italic'>Nat. Photon</em>. <b>5</b> (3), 176 (2011).</li><li>Nomura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Room-temperature+fabrication+of+transparent+flexible+thin-film+transistors+using+amorphous+oxide+semiconductors.">Room-temperature fabrication of transparent flexible thin-film transistors using amorphous oxide semiconductors.</a> <em style='font-style: italic'>Nature</em>. <b>432</b> (7016), 488 (2004).</li><li>Pirkle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+chemical+residues+on+the+physical+and+electrical+properties+of+chemical+vapor+deposited+graphene+transferred+to+SiO2.">The effect of chemical residues on the physical and electrical properties of chemical vapor deposited graphene transferred to SiO2.</a> <em style='font-style: italic'>Applied Physics Letters</em>. <b>99</b> (12), 122108-122110 (2011).</li><li>Chae, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+large-area+graphene+layers+on+poly-nickel+substrate+by+chemical+vapor+deposition:+wrinkle+formation.">Synthesis of large-area graphene layers on poly-nickel substrate by chemical vapor deposition: wrinkle formation.</a> <em style='font-style: italic'>Advanced Materials</em>. <b>21</b> (22), 2328-2333 (2009).</li><li>Zhu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+electronic+transport+in+graphene+wrinkles.">Structure and electronic transport in graphene wrinkles.</a> <em style='font-style: italic'>Nano Letters</em>. <b>12</b> (7), 3431-3436 (2012).</li><li>Paronyan, T. M., Pigos, E. M., Chen, G., Harutyunyan, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+ripples+in+graphene+as+a+result+of+interfacial+instabilities.">Formation of ripples in graphene as a result of interfacial instabilities.</a> <em style='font-style: italic'>ACS Nano</em>. <b>5</b> (12), 9619-9627 (2011).</li><li>Stadermann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+large-area+free-standing+ultrathin+polymer+films.">Fabrication of large-area free-standing ultrathin polymer films.</a> <em style='font-style: italic'>Journal of Visualized Experiments : JoVE</em>.  (100), e52832 (2015).</li><li>Zhou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Nanoporous+Alumina+Ultrafiltration+Membrane+with+Tunable+Pore+Size+Using+Block+Copolymer+Templates.">Fabrication of Nanoporous Alumina Ultrafiltration Membrane with Tunable Pore Size Using Block Copolymer Templates.</a> <em style='font-style: italic'>Advanced Functional Materials</em>. <b>27</b> (34), 1701756 (2017).</li><li>Meitl, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+printing+by+kinetic+control+of+adhesion+to+an+elastomeric+stamp.">Transfer printing by kinetic control of adhesion to an elastomeric stamp.</a> <em style='font-style: italic'>Nature Materials</em>. <b>5</b> (1), 33 (2006).</li><li>Suk, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+CVD-grown+monolayer+graphene+onto+arbitrary+substrates.">Transfer of CVD-grown monolayer graphene onto arbitrary substrates.</a> <em style='font-style: italic'>ACS Nano</em>. <b>5</b> (9), 6916-6924 (2011).</li><li>Chen, Y., Gong, X. L., Gai, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+Challenges+in+Transfer+of+Large-Area+Graphene+Films.">Progress and Challenges in Transfer of Large-Area Graphene Films.</a> <em style='font-style: italic'>Advanced Science</em>. <b>3</b> (8), 1500343 (2016).</li></ol>

<p class="jove_content">The authors have nothing to disclose.</p>

<p class="jove_content">While many of the steps listed in this protocol are crucial for the success of the thin film transfer, the nature of the custom-designed 3D printed drain chamber allows for broad flexibility, according to the user's specific requirements. For example, if the receiver substrate has a larger diameter than the 25-mm-diameter AAO discs utilized in this study, the drain chamber can be appropriately modified to fit the new specifications. However, there are certain aspects of the protocol that are necessary to ensure effective...

<p class="jove_content">Thin film and nanomembrane-based devices have recently garnered wide interest due to their potential use in a broad range of applications, ranging from flexible photovoltaics and photonics, foldable displays, and wearable electronics<sup class="xref">1</sup><sup>,</sup><sup class="xref">2</sup><sup>,</sup><sup class="xref">3</sup>. A requirement for the fabrication of these various types of devices is the transfer of thin films to the surfaces of arbitrary substrates, which remains challenging due to the fragility of these films and the frequent production of macroscale defect structures, such as wrinkles, cracks, and tears, within the films after transfer<sup class="xref">4</sup><sup>,</sup><sup class="xref">5</sup><sup>,</sup><sup class="xref">6</sup><sup>,</sup><sup class="xref">7</sup>. Manual transfer by hand, tweezers, and wire loops are common methods of thin film transfer, but inevitably result in structural incongruities and plastic deformation<sup class="xref">8</sup><sup>,</sup><sup class="xref">9</sup>. Various types of thin film transfer methodologies have been explored such as: 1) polydimethylsiloxane (PDMS) stamp transfer, which involves the use of an elastomeric stamp to obtain the thin film from the donor substrate and subsequently transfer to the receiving substrate<sup class="xref">10</sup>, and 2) sacrificial layer transfer<sup class="xref">11</sup>, in which an etchant is used to selectively dissolve a sacrificial layer between the support substrate and the thin film, thereby lifting off the thin film. However, these techniques alone do not necessarily allow for thin film transfer without incurring damage to or defect formation within the thin films<sup class="xref">12</sup>.</p>
<p class="jove_content">Here, we present a novel, low-cost, and generalizable facile method based on sacrificial layer lift-off and meniscus-guided transfer within a custom-designed, 3D-printed drain chamber system, to mechanically place block copolymer (BCP) thin films onto the centers of porous substrates such as anodized aluminum oxide (AAO) discs with little-to-no incurred macroscale defect structures, such as wrinkles, tears, and cracks. In the present context, these transferred thin films can then be used as devices in water filtration studies, potentially after sequential infiltration synthesis (SIS) processing<sup class="xref">9</sup>. Image analysis of transferred films obtained from optical microscopy show that the meniscus-guided, drain-chamber system provides smooth, robust, and wrinkle-free samples. In addition, the images also demonstrate the system&#39;s ability to reliably place the thin film membranes onto the centers of the receiving substrates. Our results have significant implications for any type of device application requiring the transfer of thin film structures onto the surfaces of arbitrary porous substrates.</p>

<table><tbody><tr><td>35% sodium polyacrylic acid solution</td><td>Sigma Aldrich</td><td>9003-01-4&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Amicon Stirred Cell model 8010 10mL</td><td>Millipore</td><td>5121</td><td></td></tr><tr><td>Anodized aluminum oxide, 0.2u thickness, 25mm diameter</td><td>Sigma Aldrich</td><td>WHA68096022</td><td></td></tr><tr><td>o ring neoprene 117</td><td>Grainger</td><td>1BUV7</td><td></td></tr><tr><td>Objet500 Connex3 3D Printer</td><td>Stratasys</td><td></td><td></td></tr><tr><td>Onshape 3D software</td><td>onshape</td><td></td><td></td></tr><tr><td>Polylactic acid filament</td><td>Ultimaker</td><td></td><td></td></tr><tr><td>ultimaker3 3d filament printer</td><td>Ultimaker</td><td></td><td></td></tr><tr><td>Vero Family printable materials</td><td>Stratasys</td><td></td><td></td></tr></tbody></table>

procedure for the transfer of polymer films onto porous substrates with minimized defects

<p class="jove_content">The fabrication of devices containing thin film composite membranes necessitates the transfer of these films onto the surfaces of arbitrary support substrates. Accomplishing this transfer in a highly controlled, mechanized, and reproducible manner can eliminate the creation of macroscale defect structures (e.g., tears, cracks, and wrinkles) within the thin film that compromise device performance and the usable area per sample. Here, we describe a general protocol for the highly controlled and mechanized transfer of a polymeric thin film onto an arbitrary porous support substrate for eventual use as a water filtration membrane device. Specifically, we fabricate a block copolymer (BCP) thin film on top of a sacrificial, water-soluble poly(acrylic acid) (PAA) layer and silicon wafer substrate. We then utilize a custom-designed, 3D-printed transfer tool and drain chamber system to deposit, lift-off, and transfer the BCP thin film onto the center of a porous anodized aluminum oxide (AAO) support disc. The transferred BCP thin film is shown to be consistently placed onto the center of the support surface due to the guidance of the meniscus formed between the water and the 3D-printed plastic drain chamber. We also compare our mechanized transfer-processed thin films to those that have been transferred by hand with the use of tweezers. Optical inspection and image analysis of the transferred thin films from the mechanized process confirm that little-to-no macroscale inhomogeneities or plastic deformations are produced, as compared to the multitude of tears and wrinkles produced from manual transfer by hand. Our results suggest that the proposed strategy for thin film transfer can reduce defects when compared to other methods across many systems and applications.</p>

<p class="jove_content" fo:keep-together.within-page="1">The BCP membrane samples were fabricated according to the previously described procedure<sup class="xref">9</sup>. The samples were placed onto the lip of the loading arm of the 3D-printed transfer tool (<strong class="xfig">Figure 1</strong>, left) and subsequently lowered, with a laboratory jack, onto the entrance ramp of the 3D-printed drain chamber tool (<strong class="xfig">Figure 1</strong>, right). A sacrificial layer of poly(acrylic acid) (PAA) between the BCP membrane and underly...

Watch this Scientific Journal Video about Procedure for the Transfer of Polymer Films Onto Porous Substrates with Minimized Defects at JoVE.com

Procedure for the Transfer of Polymer Films Onto Porous Substrates with Minimized Defects

<p class="jove_content">We present a procedure for highly controlled and wrinkle-free transfer of block copolymer thin films onto porous support substrates using a 3D-printed drain chamber. The drain chamber design is of general relevance to all procedures involving transfer of macromolecular films onto porous substrates, which is normally done by hand in an irreproducible fashion.</p>

The fabrication of devices containing thin film composite membranes necessitates the transfer of these films onto the surfaces of arbitrary support ...

procedure-for-transfer-polymer-films-onto-porous-substrates-with

Research

JoVE Journal

Engineering

6.5K Views.  University of Chicago. We present a procedure for highly controlled and wrinkle-free transfer of block copolymer thin films onto porous support substrates using a 3D-printed drain chamber. The drain chamber design is of general relevance to all procedures involving transfer of macromolecular films onto porous substrates, which is normally done by hand in an irreproducible fashion.

Video: Procedure for the Transfer of Polymer Films Onto Porous Substrates with Minimized Defects

Column Chromatography

<p><strong>Column Chromatography</strong></p>
<p>Multiple techniques exist to purify and separate compounds in an organic chemistry laboratory. One of the most reliable separation techniques for microscale separations, where less than 10 grams of the compound is available for analysis, is column chromatography. This technique separates compounds in a mixture based on their physical properties, such as their solubility, polarity, hydrophobicity, size, and charge.</p>
<h4><strong>Chromatography Basics</strong></h4>
<p>The main components of any chromatography system are the stationary phase, the mobile phase, and the sample mixture to be analyzed. In column chromatography, the stationary phase is typically microscale beads, which are packed uniformly in a vertical column. A continuous flow of the mobile phase, also known as the solvent, is added to the top of the column, which flows through the stationary phase via gravity or at a controlled flow rate by a pump.</p>
<p>The sample is dissolved in a small amount of the solvent and then added to the top of the column. More solvent is added to force the sample to flow through the stationary phase. The compounds in the mixture partition between the mobile phase and the stationary phase based on their differing properties and form discrete bands.</p>
<p>Compounds with strong interactions with the stationary phase will move slower than compounds with weak interactions with the stationary phase. Thus, compounds with weak interactions will exit the column, or elute, earlier than those with strong interactions. The strength of a compound&#8217;s interaction with the stationary phase is defined by the retardation factor (R<sub>f</sub>), which is the ratio of the distance traveled by a component to that traveled by the mobile phase. The retardation factor is determined by first performing thin layer chromatography.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/63/63_Concepts_1.jpg" /></p>
<p>A high retardation factor value indicates that the sample interacts strongly with the stationary phase and takes a long time to elute. A low retardation factor value indicates that the sample does not interact with the stationary phase very strongly and elutes more quickly. Since compounds partition into individual bands and elute from the column at different times due to their different R<sub>f</sub> values, they can be isolated individually by collecting the solvent from under the column in fractions.</p>
<h4><strong>Column Chromatography Considerations</strong></h4>
<p>A chromatography column is a vertically oriented glass or plastic tube. The size of the column can range from a few centimeters (such as a Pasteur pipette) to meters (such as with industrial columns). The diameter of the column depends on the amount of sample to be separated, whereas the length of the column depends on the difficulty of separation. The most efficient partitioning into discrete bands requires thin sample layers; therefore, determining the appropriate diameter is a vital step. Loading too much sample volume relative to the column&#8217;s diameter will create broader and less defined bands than the same volume of sample in a column with a larger diameter.</p>
<p>When choosing the length of the column, the R<sub>f</sub> values of the components must be considered. Components with similar retardation factors may be challenging to separate and require a longer column to resolve the individual bands. Compounds with very different retardation factors may not need a long column, as they should partition easily. When selecting the stationary phase, it is important to choose one that results in different retardation factors for each component.</p>
<p>Preparing the column is the most critical part of running a chromatography column. The mass of the stationary phase packed in the column should be at least 20x that of the sample. If the column does not have a porous disk at the bottom, it should be packed with cotton and a thin layer of sand. This prevents the loss of the stationary phase through the exit of the column.</p>
<p>There are two methods to pack the column: the dry packing method and the slurry method. In the dry method, the stationary phase is transferred into the column in its powder form. The column is then washed several times with the mobile phase/solvent to ensure that the solvent penetrates every part of the silica gel column. This method, if not done correctly, can increase the probability of dry patches, channels, and air bubbles forming in the column. These defects will affect the ability of the column to partition components of a mixture.</p>
<p>The slurry method provides a more uniform packed column that does not contain air bubbles, dry areas, and channels. For this method, the stationary phase is mixed with a solvent to form a consistent slurry. The slurry is then transferred to the column while tapping at the sides to get rid of any air bubbles formed. The stopcock of the column, if there is one, should be open during this step to allow the solvent to drain.</p>
<h4><strong>Silica Gel Chromatography</strong></h4>
<p>Many different properties are exploited to separate a mixture of compounds, such as size, charge, and hydrophobicity. One property used to separate compounds in organic chemistry is polarity. For this, silica gel and alumina gel are the most commonly used stationary phases. Silica gel is extremely polar and forms strong dipole-dipole interactions with polar compounds. In addition, silica gel can form hydrogen bonds with the analyte due to the presence of -OH groups on its surface.</p>
<p>Components of the mixture that are most polar bind strongly to the silica gel and travel slowly through the column, while non-polar components are more soluble in the mobile phase and travel quickly through the column. Thus, it is essential that the components of the mixture have different polarities. Components that interact strongly with the stationary phase are eluted by flowing a polar mobile phase through the column.</p>
<h4><strong>References</strong></h4>
<ol>
	<li style="font-size:17px">Shuler, M.L., Kargi, K., DeLisa, M. (2017). <em>Bioprocess Engineering, Basic Concepts.</em> Upper Saddle River, NJ: Prentice Hall.</li>
	<li style="font-size:17px">Harris, D.C. (2015).<em> Quantitative Chemical Analysis.</em> New York, NY:<em> W.H. Freeman and Company.</em></li>
</ol>

Column Chromatography: Experimental Setup and Separation - Concept

Column Chromatography
Multiple techniques exist to purify and separate compounds in an organic chemistry laboratory. One of the most reliable separation ...

Education

JoVE Lab Manual

Chemistry

Hydrolysis of an Ester

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you get started, put on a lab coat, safety glasses, and nitrile gloves. Ensure that the lab is equipped with enough lab coats, splash goggles, and gloves for all of the students.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 1 L of 3 M NaOH. Measure 120 g of NaOH pellets, and transfer the pellets to a 1-L volumetric flask. Add deionized water to the 1-L line. Place a stir bar in the flask and mix the solution until the pellets are fully dissolved.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Once the solution is homogeneous, transfer the solution to a glass bottle, cap it, and label it. Place the bottle in a central hood so that students have access to it.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Prepare 1 L of 1 M CaCl<sub>2</sub> solution. Weigh 110.98 g of CaCl<sub>2</sub>, and transfer it to a 1-L volumetric flask. Fill the flask to the marked line with deionized water, add a stir bar, and mix the solution on a stir plate until the CaCl<sub>2</sub> is fully dissolved.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Once the solution is homogeneous, transfer it to a clean glass bottle, cap it, and label it. Store the solution in a central hood.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Prepare at least 1 L of 3 M HCl, which will be used to neutralize any excess sodium hydroxide at the end of the experiment.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Obtain 5 silicone soap molds and leave them in a common area. Obtain at least five different fragrance oils and five powder soap dyes in a variety of colors, and set them near the molds.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place a container of commercial liquid soap in a common area. Set coconut oil next to the balance making sure there are clean spatulas and weighing boats.</div>
		</li>
		<li data-sub-note-item="1-10">
		<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" height="224" 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;Stir bar</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;Package of pH paper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;pH meter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL plastic squeeze bottle of DI water</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;50-mL volumetric flasks</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;100-mL glass graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;6&#160;&#160;&#160;&#160;50-mL glass graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Glass vials</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small spoon</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small spatula</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

Hydrolysis of an Ester: Preparation of Soap - Prep

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

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

Lab Manual Sub Articles

Recrystallization

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Recrystallization of Acetanilide<button class="expand">Expand</button>
	<p>In this part of the lab, you&#39;ll purify acetanilide by recrystallization from an aqueous solution. Acetanilide is moderately soluble in boiling water, but it&#39;s much less soluble in room temperature and cold water. Aniline, a potential impurity, is much more soluble in room temperature water. Thus, you&#39;ll first make a saturated solution of acetanilide in boiling water. Then, you&#39;ll slowly cool the solution to room temperature.</p>
	<p>As the acetanilide&#39;s solubility decreases, it will slowly form flat colorless or white crystals. Water-soluble impurities will remain in solution. You will then cool the solution to further decrease the solubility of acetanilide. Finally, you&#39;ll recover the crystals with vacuum filtration and measure the melting point of your crystals.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before you start the lab, put on a lab coat, safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">To begin, bring a 125-mL Erlenmeyer flask and your lab notebook to the analytical balance to obtain your acetanilide.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Measure 2 g of acetanilide in a tared weighing boat and record the precise mass in your lab notebook. Add the acetanilide to the flask and bring it back to your fume hood.
		<h4><strong>Table 1: Recrystallization of acetanilide</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Compound</strong></td>
					<td><strong>Mass (g) </strong></td>
					<td><strong>Melting point (&#176;C) </strong></td>
				</tr>
				<tr>
					<td>Acetanilide</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Seed crystal (if used)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Recrystallized acetanilide<br />
					(<em>subtract seed crystal if used</em>)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td><strong>Percent recovery</strong></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/58/Table_1._Recrystallization_of_acetanilide.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Then, measure 30 mL of deionized water and carefully pour it into the flask of acetanilide.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Place the flask on a stirring hotplate, add a stir bar, and set the hotplate to medium heat and stirring. Let the mixture boil for 4 &#8211; 5 min to dissolve the acetanilide.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">If you see solid acetanilide after the solution has boiled for 5 min, measure another 10 mL of deionized water. Use a Pasteur pipette to add water dropwise until either the solid dissolves or you&#39;ve added all 10 mL.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Let the solution boil for at least a minute after each milliliter before adding more water.<strong> Note:</strong> It&#39;s important to add water slowly so that you don&#39;t cool the solution too much or add more water than you need.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">If you still see some solid after that, those are impurities that you must remove by hot filtration. If you do not see solid, continue the lab at step 14.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">To perform hot filtration, place about 15 mL of deionized water and one or two boiling chips in a 50-mL beaker and start heating it on a hotplate.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Clamp another 125-mL Erlenmeyer flask on a hotplate and place a wide stem or stemless glass funnel in the flask. Fold a large piece of circular filter paper in quarters, place it point down in the funnel, and open it into a cone.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once the water boils, use a pipette to wet the filter paper with hot water. Then, use tongs to pick up the flask of acetanilide solution and pour the hot liquid into the funnel, being careful not to let it overflow. <strong>Note:</strong> If the stir bar falls into the funnel, retrieve it with tweezers and rinse it with hot water.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Then, rinse the inside of the first flask with a few milliliters of hot water, pour the contents into the funnel, and dissolve any crystals that form with hot water.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Once the funnel is empty, use tongs to move it to the first flask. Carefully unclamp the second flask. You can now continue with the lab as usual.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Once there is no visible solid in your hot acetanilide solution, turn off the heat and stir motor and use tongs to place the flask on the floor of the hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Leave the solution undisturbed as it cools to room temperature, which usually takes at least 15 min. Moving or shaking the flask will make it harder for crystals to form.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">While you wait, place a small amount of the starting acetanilide material in a weighing boat and bring it to the melting point apparatus.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Pack a capillary tube with the acetanilide and measure and record its melting point.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Once the solution cools, look for crystals without moving the flask too much. If you don&#39;t see any, use a glass rod to gently scratch the inside of the flask under the solution. It&#39;s easier for crystals to start to form on scratched or rough surfaces.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">If crystals still haven&#39;t formed after 10 more min, ask your instructor for a seed crystal of high purity acetanilide. Measure and record the mass of the seed crystal, and then drop it into the room temperature acetanilide solution. Crystal growth will then occur on the seed crystal.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Once you see several crystals in the flask, prepare two ice baths in 600-mL beakers. One ice bath should nearly fill the beaker, and the other should occupy about half of the beaker.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Measure 5 mL of deionized water and place it in the full ice bath. Confirm that the flask is cool to the touch and that several crystals have formed before gently placing it in the half-full ice bath. Leave the flask undisturbed to improve crystal growth.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Check the crystal growth progress every 15 min. Once it looks like most of your acetanilide has crystallized, or you don&#39;t see much crystal growth over a 15-min period, set up for a vacuum filtration using a 125- or 250-mL filter flask.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Wet the filter paper with 1 mL of ice-cold water. Then, start the vacuum and pour the contents of the flask into the B&#252;chner funnel of the vacuum filtration setup.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Scrape crystals from the flask into the funnel using a glass rod or flexible spatula and rinse the remaining crystals into the funnel with about 1 mL of cold deionized water.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Then, stop the vacuum, break the seal, and pour the rest of the cold deionized water into the funnel. Gently stir the crystals with the glass rod for a few seconds to wash them, being careful not to tear the filter paper.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Turn on the vacuum to drain the funnel. Then, stop the vacuum, break the seal, and empty the filtrate into a waste beaker. Turn the vacuum back on, and leave the washed crystals in the funnel while the vacuum pump pulls air through them until they are dry, which usually takes about half an hour. You&#39;ll start working on your next recrystallization while you wait.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Recrystallization of <em>trans</em>-Cinnamic Acid<button class="expand">Expand</button>
	<p>In the second half of the lab, you&#39;ll recrystallize <em>trans</em>-cinnamic acid, which we&#39;ll just call cinnamic acid. Cinnamic acid is soluble in ethanol and nearly insoluble in water. So, you&#39;ll use a mixture of 95% ethanol and 5% water so that cinnamic acid is still soluble at high temperatures but less soluble at low temperatures.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">To get started, fill a 5- or 10-mL graduated cylinder with deionized water and bring it to your hood.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Then, weigh 0.5 g of cinnamic acid, record its mass, and place it in a 25-mL Erlenmeyer flask.
		<h4><strong>Table 2: Recrystallization of<em> trans</em>-Cinnamic acid</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Compound</strong></td>
					<td><strong>Mass (g) </strong></td>
					<td><strong>Melting point (&#176;C) </strong></td>
				</tr>
				<tr>
					<td><em>trans</em>-Cinnamic acid</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Seed crystal (if used)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Recrystallized<em> trans</em>-cinnamic acid<br />
					<em>(subtract seed crystal if used)</em></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td><strong>Percent recovery</strong></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/58/Table_2._Recrystallization_of_trans-Cinnamic_acid.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Next, obtain 4 mL of 95% ethanol, and pour it into the flask containing cinnamic acid. Place the flask on a hotplate set to medium heat and stir the solution with a glass rod until the cinnamic acid dissolves, which usually takes 7 &#8211; 10 min.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Then, turn off the heat and use a Pasteur pipette to add 5 drops of deionized water to the solution. This reduces cinnamic acid solubility. <strong>Note:</strong> If you don&#39;t see precipitation, add up to 5 more drops of water one by one. Stop adding water if you see solids starting to form.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Then, remove the flask from the hotplate and let it cool to room temperature undisturbed.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">While you wait, get a small amount of the starting cinnamic acid and measure its melting point.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Once the flask has cooled to room temperature, inspect it for crystals. If you don&#39;t see any, gently scratch the inside of the flask under the solution with the glass rod and wait for 10 min. <strong>Note:</strong> If that still doesn&#39;t work, get a seed crystal of cinnamic acid from your instructor. Remember to weigh the seed crystal and record its mass before you add it to your solution.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Once the flask is cool to the touch and contains several crystals, remake the ice baths with one ice bath shallow enough that the 25-mL flask will not be completely submerged.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Refill the graduated cylinder with deionized water. Place the flask of cinnamic acid crystals in the shallow ice bath and the water-filled graduated cylinder in the other ice bath. Let the crystals grow for at least 15 min.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Your acetanilide crystals should be dry by now, so measure their mass and melting point while you wait for your cinnamic acid to finish crystallizing. Turn off the vacuum and break the vacuum seal.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Gently place the B&#252;chner funnel in a clean beaker and bring it to the balance. Measure and record the mass of your pure acetanilide crystals. Remember to subtract the mass of the seed crystal if you used one.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Then, bring your crystals to the melting point apparatus and measure and record the melting point.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Now, place a clean, dry B&#252;chner funnel with a new piece of filter paper in the filter flask and check on your cinnamic acid. Once it looks like most of the cinnamic acid has crystallized, wet the filter paper with cold deionized water.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Start the vacuum and pour the contents of the cinnamic acid flask into the funnel. Scrape the remaining crystals out of the flask with the glass rod and rinse the flask with about 1 mL of cold water.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Now, turn off the vacuum, break the seal, and pour the rest of the cold water into the funnel. Gently stir the crystals with the glass rod to wash them, being careful not to damage the filter paper.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Turn on the vacuum to drain the funnel. Leave the vacuum running until the cinnamic crystals are dry.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Place the B&#252;chner funnel in a beaker and bring it to the balance. Measure and record the mass of your pure cinnamic acid crystals. Remember to subtract the mass of the seed crystal if you used one. Then, measure the melting point of your recrystallized cinnamic acid.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Now, dispose of your acetanilide and cinnamic acid in the designated solid waste container. Flush the filtrate down the drain with running water and pour leftover ice and water into the sink.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Clean your glassware using your usual methods and put away your other lab equipment.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Lastly, discard your used pipettes in the glass waste and throw out used filter paper and other trash in the lab trash container.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">First, calculate the percent recovery for each compound. We expect that some of each compound stayed in solution, so these percentages will be below the reported purity of your starting material.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Now, look at the differences in melting points between the starting material and the crystals. For each compound, the less pure starting material has a lower melting point and a broader range than the recrystallized compound.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Lastly, compare your results to the literature values. The melting point of acetanilide is 114 &#176;C, and the melting point of <em>trans</em>-cinnamic acid is 133 &#176;C. The melting points of your crystals should span 1 &#8211; 2 &#176;C and should be at or just below those values.</div>
		</li>
	</ul>
	</li>
</ol>

Recrystallization of Acetanilide and trans-Cinnamic Acid - Procedure

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

	Recrystallization of AcetanilideExpand
	In this part of ...

Basic Organic Chemistry Techniques

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Synthesis of Benzoic Acid<button class="expand">Expand</button>
	<p>In this lab, you&#39;ll learn several important techniques that you&#39;ll use throughout this class. This includes measuring the mass or volume of reactants and products, methods of heating and cooling reaction mixtures, proper selection and handling of glassware, and separating solids and liquids by vacuum filtration. First, you&#39;ll synthesize benzoic acid by protonating sodium benzoate with hydrochloric acid, or HCl, using water as the solvent. Sodium benzoate is very soluble in water, but benzoic acid is not, so you can filter the solid product out of the solution.</p>
	<p>You&#39;ll use vacuum filtration, which is used for powdery solids, to filter out the benzoic acid. Benzoic acid is less soluble in water at lower temperatures, so you&#39;ll collect more product if you cool the reaction solution before you filter it. When you do this lab, remember that HCl is toxic and a strong acid. It&#39;s always best to work with strong acids and organic solvents in a fume hood. If you spill any HCl, follow your lab&#39;s procedures for neutralizing and cleaning up strong acids.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on a lab coat, safety glasses, and nitrile gloves. Change your gloves if you get acid or organic solvent on them.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Bring a clean 50-mL beaker and your lab notebook to the balance, and then tare a clean empty weighing boat on the balance.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use a clean spatula to measure out ~2 g of sodium benzoate.<strong> Note:</strong> Stay between 1.9 and 2.1 g.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Dispose of any extra sodium benzoate into the appropriate waste container. Close the bottle of sodium benzoate when you&#39;re done with it.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Write down the mass of sodium benzoate and pour it into your beaker. Clean the spatula with a lab wipe so that it can be used again and return to your fume hood.
		<h4><strong>Table 1: Reaction yield of benzoic acid</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Mass of sodium<br />
					benzoate (g)</strong></td>
					<td style="text-align: center;"><strong>Moles of sodium<br />
					benzoate</strong></td>
					<td style="text-align: center;"><strong>Volume of<br />
					HCl (mL)</strong></td>
					<td style="text-align: center;"><strong>Moles of<br />
					HCl</strong></td>
					<td style="text-align: center;"><strong>Moles of benzoic<br />
					acid</strong></td>
					<td style="text-align: center;"><strong>Mass of benzoic<br />
					acid (g)</strong></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td><strong>Theoretical yield</strong></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">-</td>
					<td style="text-align: center;">-</td>
					<td style="text-align: center;">-</td>
					<td style="text-align: center;">-</td>
					<td><strong>Percent yield</strong></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/56/Table_1._Reaction_yield_of_benzoic_acid.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Get a clean 10-mL graduated cylinder and measure 10 mL of deionized water. Pour the water into the beaker of sodium benzoate. Stir the solution with a glass rod until the sodium benzoate has completely dissolved.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Obtain a clean 5-mL graduated cylinder and measure 5 mL of 3 M HCl. <strong>Note: </strong>If you pour too much, use a Pasteur pipette to transfer the excess to a small beaker labeled &#8216;acid waste&#8217;. Close the bottle and put it back when you&#39;re done.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use a clean Pasteur pipette to draw up a small amount of HCl from your graduated cylinder. Start adding the HCl to the sodium benzoate solution at a rate of about one drop every 6 seconds or 10 drops/min. Stir the solution frequently with the glass rod. This should take about 10 min. <strong>Note:</strong> As you add more HCl, you&#39;ll start to see benzoic acid precipitating from the solution.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">When you are done, fill a 600-mL beaker about halfway with crushed ice and add tap water to just fill the spaces between the ice to make an ice bath.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Place the ice bath by a lab stand in your fume hood and use a medium clamp to hold the reaction beaker in the bath. Make sure that the surface of the solution is below the ice.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Clamp a thermometer in the reaction solution.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">While the reaction cools, obtain 25 mL of deionized water in a 50-mL graduated cylinder. Place the graduated cylinder in a 600-mL beaker and pack ice around it to cool the water.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Once the reaction solution reaches 10 &#176;C, it&#39;s ready to be filtered. Set up another lab stand and obtain a clean 250-mL filter flask. Clamp the flask upright on the lab stand.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Ensure that your vacuum tubing is connected to the vacuum source, and then carefully push the free end of the tubing onto the barbed arm of the flask.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Place a rubber adapter and a B&#252;chner funnel in the mouth of the flask. Confirm that the reaction solution has reached 10 &#176;C and remove the thermometer from the beaker.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Obtain a piece of circular filter paper and place it in the B&#252;chner funnel so that the small holes are completely covered.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Get a new Pasteur pipette, and use a few drops of cold deionized water to wet the filter paper. <strong>Note: </strong>This helps keep the filter paper in place when you pour the solution into the funnel.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Open the vacuum line part way to reduce the pressure in the flask.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Remove the beaker from the ice bath, stir the solution well with the glass rod until the solid is swirling around in the liquid, and then slowly pour it onto the filter paper. <strong>Note:</strong> If the liquid does not move quickly through the funnel, make sure that there are no gaps between the funnel, adapter, and flask, and then gradually open the vacuum until the liquid rapidly flows into the flask.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Use a clean Pasteur pipette to rinse the inner walls of the beaker with ~10 mL of cold deionized water and pour the rinse into the funnel. Pour the rest of the cold deionized water over the benzoic acid on the filter to wash it. <strong>Note:</strong> This is an important step that removes impurities, side products, and unreacted compounds.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Leave the benzoic acid in the funnel with the vacuum running until it is completely dry. <strong>Note: </strong>This usually takes ~2 h, so let&#39;s continue to the second half of the lab in the meantime.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Refluxing Tetrahydrofuran (THF) and Measuring Product Mass<button class="expand">Expand</button>
	<p>While your benzoic acid dries, you&#39;ll practice refluxing tetrahydrofuran, or THF, which has a boiling point of 66 &#176;C. THF is volatile, flammable, and an irritant, so avoid letting it touch your skin and always work with it in a fume hood. Refluxing is a technique that constantly condenses the vapor and returns it to the flask. This allows reactions to be performed at high temperatures, such as at the solvent&#39;s boiling point, without losing the solvent to evaporation.</p>
	<p>Refluxing is achieved with a special piece of glassware called a condenser, which is attached directly to your flask. The condenser is cooled with water flowing through the outer chamber. As vapor rises through the condenser, it loses heat to the cool walls, condenses, and drips back into the flask.</p>
	<p>Refluxing is usually performed in a round-bottom flask. These flasks must be heated in a heating bath or with specialized equipment, but they provide more even heating than an Erlenmeyer flask or beaker as a result. Water baths are typically used for experiments performed below 80 &#8211; 100 &#176;C.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-4">
		<div class="lab-step">Set up the glassware and equipment for reflux. Place a lab jack by your available lab stand and set a stirring hotplate on top of it. Clamp a 50-mL round-bottom flask at least 12 cm above the hotplate.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Bring a 50-mL graduated cylinder to the solvent hood and measure 25 mL of THF. If you pour too much, label a small beaker &#8216;organic waste&#8217; and pipette the excess into that. Remember to cap the bottle when you&#39;re done.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Carefully pour the THF into the round-bottom flask, clean up any spills with a lab wipe or paper towel, and add a small oblong stir bar to the flask.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Position a medium clamp ~20 cm above the flask. Obtain a condenser and apply vacuum grease to the lower ground-glass joint.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Fit the condenser into the flask and rotate them in opposite directions until the vacuum grease is evenly spread between the two ground-glass surfaces.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Clamp the condenser in place and use a lab wipe to remove excess grease from the connection between the two pieces of glassware.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Attach rubber tubing to each of the ports on the condenser. Place the free end of the tubing connected to the top port, which is the outlet, in the drain. Connect the free end of the tubing from the bottom port, which is the inlet, to the waterline.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Fill a 250-mL beaker with ~200 mL of tap water and place this water bath on the hotplate. Use the lab jack to raise the bath until the water level is about 2/3 of the way up the spherical part of the flask. This will ensure the THF heats evenly.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Secure a thermometer (range 0 &#8211; 100 &#176;C) in the bath using the thermometer clamp.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Slowly open the water tap, watch the condenser for leaks as it fills, and confirm that the water is flowing into the drain.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Slowly turn up the magnetic stirrer until the THF is stirring well. If the stir bar bounces around, turn down the stir motor and try again more slowly.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Start heating the water bath to about 70 &#176;C until the THF starts to boil. <strong>Note: </strong>You&#39;ll see vapor condensing in the lower part of the condenser).</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Monitor the refluxing THF for 10 min. <strong>Note:</strong> Turn down the heat if the bath goes above 70 &#176;C or if vapor leaves the condenser.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">After 10 min, remove the thermometer and turn off the heat and the stir motor. Carefully lower the lab jack until the flask is clear of the water bath. Wait at least 10 more min for the setup to cool, then unplug and remove the hotplate.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Turn off the water tap, unclamp the condenser, and carefully detach it from the flask. Tilt the condenser until most of the water has flowed through the outlet tubing into the drain. Then, disconnect the inlet tubing from the condenser and let the remaining water flow into the drain. Do the same for the outlet tubing.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Use lab wipes to remove the vacuum grease from the condenser and the flask, then retrieve the stir bar from the flask.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Bring the flask to the waste hood and empty it into the non-halogenated organic waste container. Pour any excess HCl or THF into the acid and organic wastes, respectively. Empty the ice and water baths into the sink and rinse the beakers with tap water.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Clean most of your other glassware while the benzoic acid from earlier finishes drying according to your lab&#8217;s standard practices. Usually, you&#39;ll rinse your glassware both with an organic solvent, like acetone, and with detergent and deionized water. <strong>Note:</strong> Your lab may have specific procedures for glassware used with acids. If so, follow that procedure to clean the graduated cylinder used with HCl and your acid waste beaker, if applicable.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Remove the reusable pipette bulbs and dispose of your pipettes in the glass waste container. Throw out used lab wipes and paper towels in the lab trash.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Check on your benzoic acid. If it is dry, turn off the vacuum and break the vacuum seal by disconnecting the tubing from the filter flask. Gently place the funnel in it 250-mL beaker and bring it and your lab notebook to the balance.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Tare a weighing boat and use a clean spatula to transfer the product to the weighing boat. Record the mass of your product in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Then, put the product in the lab trash and wipe off the spatula. Dispose of the filtrate in the aqueous waste container.</div>
		</li>
		<li data-sub-note-item="2-26">
		<div class="lab-step">Clean the rest of your glassware, throw out the filter paper and other waste in the lab trash, and put away your lab equipment. Lastly, clean the floor of your fume hood with a damp paper towel.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">Calculate the reaction yield for the protonation of sodium benzoate. Identify the reagent that the reaction runs out of first, which is called the limiting reagent.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Calculate the number of moles of sodium benzoate and HCl that you used. <strong>Note:</strong> You used about 0.014 moles of sodium benzoate and 0.015 moles of HCl. Thus, the maximum yield of benzoic acid is equal to the starting amount of sodium benzoate in moles.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Convert the mass of your benzoic acid to moles, divide that by the moles of sodium benzoate that you used, and multiply it by 100 to get the percent yield. <strong>Note:</strong> The recovered yield for this reaction is usually between 80% and 98% because a small amount of benzoic acid stays in solution. If the yield is above 100%, then it&#39;s likely that your product was still wet when you weighed it. Impurities, unreacted compounds, and products of side reactions can also affect the accuracy of yield calculations.</div>
		</li>
	</ul>
	</li>
</ol>

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

	Synthesis of Benzoic AcidExpand
	In this lab, you&#39;ll ...

Extraction

Extraction: Liquid-Liquid and Acid-Base Extraction - Procedure

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

	Cellulose RecoveryExpand
	Extraction and filtration can ...

Simple Distillation

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Simple Distillation<button class="expand">Expand</button>
	<p>In this lab, you will use simple distillation to separate a mixture of cyclohexane and toluene.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you start the lab, put on the appropriate personal protective equipment, including a lab coat, safety goggles, and gloves. This experiment must be conducted in a hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Assemble the distillation apparatus. Refer to the example set up that has been prepared by your instructor.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Then, set up the two stands next to each other in the hood. Place the magnetic stirrer on the base of one of the stands.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Set the heating mantle on top of the magnetic stirrer, and then plug it into the temperature controller.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Add 5 &#8211; 10 mL of sand to the heating mantle. This will allow more homogeneous heating of the round-bottom flask. Clamp the round-bottom flask to the stand above the heating mantle.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Now, obtain cyclohexane and toluene from your instructor. Measure 2 mL of cyclohexane using a volumetric pipette and dispense it into the round-bottom flask.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Then, use a clean volumetric pipette to measure 2 mL of toluene and add it to the flask.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Carefully insert a thermometer into an adapter by gently pushing and twisting it through the top.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Lightly grease the adapter with vacuum grease and insert it into the distilling head. Make sure that the thermometer bulb is below the bend of the distilling head. Then, lightly grease the joints of the distilling head.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Add a stir bar to the round-bottom flask and insert the distilling head into the round-bottom flask.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Now, clamp the condenser to the second stand with it oriented diagonally toward the side joint of the distilling head.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Connect one piece of tubing to the tap water faucet in the hood, and attach the other end to the inlet port of the condenser, which is located farthest from the distilling head.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Attach the second tubing to the outlet port, which is located closest to the distilling head, and place the other end of the tubing into the drain.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Connect the condenser to the distilling head. Then, lightly grease the joint of the condenser and attach the connecting tube. Place a 10-mL graduated cylinder below the outlet of the tube.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Gently lower the setup until the round-bottom flask is seated in the sand in the heating mantle.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Double-check all of the connections on your setup to make sure that they are tight and oriented correctly. Secure each joint with a plastic clip.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Now, slowly turn on the flow of water to the condenser. <strong>Note:</strong> Be careful not to turn on the water too quickly. If the water pressure is high, the tubing may pop off the condenser.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Turn the magnetic stirrer to the lowest setting, and set the temperature controller to heat the mantle to just above 90 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Once bubbles start forming in the liquid mixture, adjust the heat until the distillation rate is about 2 drops/min. You should also see drops forming on the thermometer bulb.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Record the temperature after every 2 drops that fall into the graduated cylinder or every minute.
		<h4><strong>Table 1: Distillation of toluene and cyclohexane</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Time (min) </strong></td>
					<td><strong>Distilled volume (mL) </strong></td>
					<td><strong>Temperature (&#176;C) </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">6</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">7</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">8</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">9</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">10</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">11</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">12</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">13</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">14</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/60/Table_1._Distillation_of_toluene_and_cyclohexane.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">When there is about 0.5 mL of the mixture remaining in the flask, which will look like a thin layer of liquid, turn off the heat and gently lift the glassware setup from the heating mantle. <strong>Note:</strong> Never heat the mixture until the flask is dry as this may cause an explosion.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Remove the stir plate with the heating mantle and set it aside.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Then, turn off the water to the condenser and allow the setup to cool completely. When it is cool, carefully disassemble the glassware.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Dispose of all solutions in the non-halogenated organic waste container. Clean your glassware with acetone followed by detergent and deionized water.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Plot the dew point temperature versus the distilled volume during the experiment. We see that drops start to fall into the collection vessel at ~ 81 &#176;C, which is the boiling point of cyclohexane. As the distillation progresses, the temperature increases to almost 111 &#176;C, which is the boiling point of toluene.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Early in the distillation, the vapor is rich in cyclohexane. As the second milliliter is distilled, the temperature increases by ~ 20 &#176;C, which tells us that the amount of toluene in the vapor is increasing significantly. The temperature stabilizes as it approaches 111 &#176;C, which tells us that the vapor is rich in toluene towards the end of the distillation.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Look at a boiling point diagram for cyclohexane and toluene, with the mole percent of cyclohexane and toluene on the x-axis and the temperature on the y-axis. The solid line denotes the bubble point, and the dashed line denotes the dew point.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Use the dew point curve to determine the composition of the vapor throughout the distillation. For example, after 0.5 mL has been distilled, the condensing vapor is about 84 &#176;C. This corresponds to 95% cyclohexane on the dew point curve.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Once you know the composition of the vapor at each point, estimate the composition of the boiling mixture throughout the distillation. At room temperature, cyclohexane and toluene have nearly the same number of moles per milliliter, so think of the percentages in terms of volume.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">To determine how much of each liquid was distilled in each step, apply each percentage to the volume of liquid distilled since the previous measurement. This tells us that we distilled 1.9 mL of cyclohexane and 1.1 mL of toluene from our mixture of 2 mL of each liquid. Thus, our distillate was 63% cyclohexane and 37% toluene, and our remaining liquid mixture was 10% cyclohexane and 90% toluene.</div>
		</li>
	</ul>
	</li>
</ol>

Simple Distillation: Separation of a Mixture of Cyclohexane and Toluene - Procedure

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

	Simple DistillationExpand
	In this lab, you will use ...

Thin-Layer Chromatography

<p>Source: Lara Al Hariri and&#160;Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Effects of Mobile Phase Composition on Compound Separation<button class="expand">Expand</button>
	<p>In this section of the lab, you will use TLC to compare how well pure hexane and a mixture of 60% hexane and 40% ethyl acetate can separate phenol, benzoic acid, and butyl phenyl ether by measuring the distance the compound traveled in each solvent.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before starting the lab, put on a lab coat, safety glasses, and nitrile gloves. Note: Hexane and ethyl acetate are both volatile, toxic, and flammable, so always work with them in a fume hood. Change your gloves if you get solvent on them, as hexane and ethyl acetate will permeate nitrile gloves within minutes.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Now, obtain a paper towel and lay it on the benchtop. Carefully pick up a TLC plate using tweezers at the very end of the plate.<strong> Note: </strong>Always hold TLC plates by the edges or at the very top with tweezers to avoid damaging or contaminating the silica gel.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Look for chips or scratches in the silica gel and get a new plate if necessary. It&#39;s fine if there is a little chipping at only one end, since that can be the top.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Place the plate on the paper towel in your fume hood, and lay a metric ruler next to it. Use a graphite pencil to gently make a small mark on the silica gel ~1 cm from the top of the plate. Write &#8216;100&#8217; on the plate above the mark.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Next, gently mark the plate 1 cm above the bottom on both sides. Carefully trace a straight line between the bottom marks to make the starting line.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Then, add 3 evenly spaced small marks to the starting line with the outer marks at least 0.5 cm from the sides of the plate. <strong>Note:</strong> It&#39;s important for the silica gel to be undamaged here, so if you scratch it with the pencil, prepare a new plate.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now, trace the outline of the TLC plate in your lab notebook and precisely copy the marks on the plate. Label the three marks as &#8216;phenol&#8217;, &#8216;benzoic acid&#8217;, and &#8216;butyl phenyl ether&#8217;. You may also want to label the marks on the plate itself.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare a second TLC plate labeled &#8216;60/40&#8217; in the same way. Now, bring the labeled plates to the common bench and locate the 1 mg/mL solutions of phenol, benzoic acid, and butyl phenyl ether in ethanol.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Dip a clean capillary into one of the three solutions. Briefly and lightly dab the capillary squarely against the intersection between the starting line and the mark for that compound. You should spot twice for each compound.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use the same capillary to spot the same solution on your second TLC plate. The spots should be no more than 2 mm in diameter. Then, discard the capillary and close the vial.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Spot the other two solutions on the plates, in the same way, using clean capillaries. Be careful not to cross-contaminate the solutions. <strong>Note</strong>: If a spot is very light or very small, wait for it to dry, and then spot the same solution on top of the dry spot. If the spot is still wet, capillary action could make it too wide and dilute.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once you have made three spots on each plate, discard the capillaries and bring the plates back to your hood. Keep the plates lying flat while they dry.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Now, label a 50-mL jar &#8216;100% hexane&#8217; and bring the jar, its cap, and a 10-mL graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Measure 2 &#8211; 3 mL of pure hexane with your graduated cylinder and pour it into the jar to fill it to a depth of 0.62 &#8211; 0.7 cm. Cap the bottle and jar and return to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Label a second 50-mL jar &#8216;60% hexane, 40% ethyl acetate&#8217; and bring it and a clean graduated cylinder to the solvent hood. Measure 2 &#8211; 3 mL of the 60/40 mixture into the jar and return to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Stand the plates next to the jars to compare the relative position of the solvent and the spots. The solvent level should be below the spots for each plate.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Then, obtain three pieces of filter paper, put a filter paper upright against the wall in each jar, and then cap the jars tightly.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Initial each plate above the mark so you can identify them later. Confirm that the filter papers in the jars have become saturated with solvent and that the spots on the plates are completely dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, open the 100% hexane jar and use tweezers to gently place the 100 plate in the jar, keeping the plate level with the solvent surface as it enters the solvent. Close the jar tightly, being careful not to jostle the plate.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Leave the jar still as the solvent travels up the plate. Development in pure hexane usually takes about 7 &#8211; 10 min. Keep an eye on the plate to make sure that the solvent doesn&#39;t reach the top.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">When the pure hexane solvent front is about 1 cm from the top of the plate, retrieve it using tweezers. Lay it on the paper towel with the silica gel facing up and quickly mark the solvent front with a line and pencil across the plate. Leave the plate lying flat as the solvent evaporates.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Place the other plate in the 60/40 eluent in the same way. Wait until the solvent front of the 60/40 plate is about 1 cm from the top, and then retrieve it and quickly mark the solvent front in the same way.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Once the 100% hexane plate is completely dry, pick it up with tweezers and bring it and a pencil to the UV lamp on the common bench.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Place the plate under the lamp and illuminate it with UV light. The plate will glow green because of a fluorescent marker in the silica gel layer. The dark spots are the compounds.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Carefully outline every spot in pencil, even if they haven&#39;t moved very far.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Then, turn off the lamp and bring your plate to the iodine development hood. Open the iodine chamber. Use tweezers to gently place the plate upright in the iodine chamber, and then cover the chamber again.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Wait 3 &#8211; 4 min for the iodine vapor to stay in the spots. Then, retrieve the plate with tweezers, close the chamber, and outline the brown and yellow spots on the plate. These spots will fade, so mark them quickly.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Visualize the dry 60/40 plate under UV light and then with iodine vapor in the same way.<strong> Note: </strong>To avoid side reactions, always check plates under UV light before exposing them to iodine.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Now, precisely copy the spots and the solvent fronts onto the corresponding drawings of the plates in your lab notebook. For each plate, measure and record the height of the solvent front relative to the starting line.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Lastly, measure the distance between the center of each spot and its starting line, and record it in your lab notebook. You can now move on to the next part of the lab.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Unknown Compound Identification<button class="expand">Expand</button>
	<p>In the second half of the lab, you&#39;ll use TLC to find out what&#39;s in an unknown analgesic. It could contain one or more of these compounds: aspirin, caffeine, and acetaminophen. These compounds are highly polar, so you&#39;ll use a premade mixture of 90% ethyl acetate and 10% hexane as the eluent.</p>
	<p>You&#39;ll run the three reference compounds and the unknown all on the same plate. Later, you&#39;ll use the Rf values of the reference compounds to identify the unknown. First, double-check which unknown material you were assigned to analyze before starting.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Now, lay a clean paper towel in the hood and obtain a new TLC plate. Prepare the top mark and the starting line the same way as before. Make four evenly spaced ticks on the plate, making sure that the outer marks are at least 0.5 cm away from the edges.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Draw an exact copy of the plate in your lab notebook and label the tick marks as &#8216;aspirin&#8217;, &#8216;acetaminophen&#8217;, &#8216;caffeine&#8217;, and &#8216;unknown&#8217;.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Now, bring your plate to the common bench. Spot solutions of aspirin, acetaminophen, caffeine, and the unknown that you were assigned on the appropriate marks using a clean capillary for each solution. <strong>Note: </strong>When you are spotting the unknown solution, don&#39;t dip the capillary too far into the solution vial to avoid clogging your capillary with insoluble material from the unknown substance.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Discard the capillaries and close the vials before bringing the plate back to your fume hood. Place the plate on a paper towel to finish drying.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Next, label a clean 50-mL screw-top jar &#8216;90% ethyl acetate, 10% hexane&#8217;, and bring it in a clean graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Place 2 &#8211; 3 mL of the 90/10 solution in the jar and return to your fume hood. Confirm that the solvent level will be below the spots on the plate. Then, place a clean filter paper upright in the jar and close it tightly.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Once the TLC plate is dry and the filter paper is saturated, carefully place the plate in the solvent. <strong>Note:</strong> It&#39;s essential to hold the plate level when you do this. If the solvent front is diagonal, the solvent distance traveled will be different for each spot, making it impossible to visually compare the unknown to the reference spots.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Wait for the solvent front to reach 1 cm from the top of the plate. Then, retrieve the plate, mark it solvent front, and let it dry while lying flat.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once the plate is dry, visualize it with UV light and in the iodine chamber, and outline the spots in pencil.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">The unknown material could be a mixture of compounds, so look carefully for spots in line with all three reference spots.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Then, precisely copy the spots onto the drawing of the plate in your lab notebook. Measure and record the height of the solvent front and the spots relative to the starting line.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">When you are finished, retrieve the solvent-saturated filter papers from the jars and let them dry in the hood.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Pour leftover solvents into the standard organic waste, wash your glassware following your lab&#39;s usual methods, and put away your lab equipment.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Throw out used TLC plates in the lab trash or with glass waste, depending on the backing material.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Lastly, once the filters are dry, throw them out along with any remaining trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">Calculate the R<sub>f</sub> values for phenol, benzoic acid, and butyl phenyl ether eluted with 100% hexane. For each compound on the TLC plate, measure the distance from the starting line to the center of its spot. Then, divide this value by the distance from the starting line to the solvent front.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Only butyl phenyl ether moved a measurable distance from the starting line, which is consistent with it being the least polar. For effective separation, the R<sub>f</sub> values should differ by 0.3 &#8211; 0.7. Thus, we can infer that phenol and benzoic acid are too polar to be separated effectively by an apolar solvent like pure hexane.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the R<sub>f</sub> values for the plate that you ran in a 60/40 mixture of hexane and ethyl acetate. Ethyl acetate is a moderately polar solvent, so the compounds traveled farther and are spaced farther apart on the plate.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">The separation order shows that benzoic acid, the most polar compound, had the highest affinity for silica gel, whereas butyl phenyl ether, the least polar compound, had the lowest affinity for silica gel. This is consistent with the assumption that less polar compounds travel farther on polar stationary phases.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Look at your third plate and calculate the R<sub>f</sub> values for aspirin, acetaminophen, and caffeine. Caffeine, the most polar complex of the three, traveled the least. Acetaminophen traveled the second farthest, while aspirin, the least polar, traveled the most.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Finally, identify the compounds in your unknown material by comparing its spot or spots to the three reference spots.</div>
		</li>
	</ul>
	</li>
</ol>

Thin-Layer Chromatography: Experimental Setup and Separation - Procedure

Source: Lara Al Hariri and&#160;Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Effects of Mobile Phase Composition on Compound ...

Stoichiometry, Product Yield, and Limiting Reactants

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Synthesis of tris(ethylenediamine)nickel chlorideExpand
	In this lab, you will combine nickel ...

Microbial and Fungal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Nutrient Agar and Bacterial Culture Plate Preparation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Add 23 g of nutrient agar to a 2,000 mL beaker containing 1 L of distilled water 4 &#8211; 5 days before the activity. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place the beaker on a hotplate and set the hotplate to high, stirring the mixture with a stirring rod.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">When the agar has dissolved into the water, use a hot pad to transfer the beaker into an autoclave and set it to run at 121 &#176;C for 15 minutes.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">When the cycle is finished, remove the beaker and allow it to cool to the touch for about 15 minutes without allowing the agar to solidify.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Then, pour the liquid nutrient agar into a Petri dish until the dish is about halfway full.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Place the lid onto the top and allow it to cool.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once the agar is solidified, seal the sides with Parafilm.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Prepare enough Petri dishes so that each student or student group will have one per bacterial type and one for the yeast, and then place these into a refrigerator for storage.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Bacteria and Yeast Plating
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To prepare for plating, place the plates under a fume hood and use a marker to draw two intersecting lines on the bottom of each plate to divide the agar into four equal-sized quadrants.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Label the bottoms of the plates with your initials and the name or initials of the bacterial species that will be plated along with the date.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">When all of the plates have been labeled, light a Bunsen burner and adjust the gas to be just high enough to sustain a blue flame, and heat the looped end of an inoculating loop in the flame of the burner for 20 seconds.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">After removing the loop from the heat, allow the metal to cool for about 15 seconds.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, place the end of the loop in one vial of bacteria, making sure that the loop is coated with bacteria.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Gently smear the loop onto one quadrant of agar in one of the plates labeled for that bacteria.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When the entire quadrant has been filled, flame sterilize the loop as previously described (steps 3 &#8211; 4), and then streak the rest of the plate following a pattern similar to this one so that the streak in the second quadrant only crosses the first streak one or two times, the third crosses the second in the same manner, and the fourth crosses the third only once.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Repeat this streak plating for the remaining two bacteria species (step 7).
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Finally, reseal the plates with Parafilm and store the cultures lid down in a 32 &#176;C incubator until the day of the activity.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">To plate the yeast culture, label the bottom of the remaining plate with the yeast strain name, date, and your name.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Using an inoculating loop, scoop up S. cerevisiae from a vial onto the agar and evenly spread the yeast across the plate.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Then, cover the plate with the lid and reseal the plate with Parafilm for upside down culture at 32 &#176;C until the day of the activity.
</div></li></ul></li></ol>

Microbial Colony and Fungal Diversity - Prep

Nutrient Agar and Bacterial Culture Plate Preparation
ExpandAdd 23 g of nutrient agar to a 2,000 mL beaker containing 1 L of distilled water 4 &#8211; 5 ...

Biology

Evolution

Macromolecules

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Identifying Macromolecules with Indicator Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">HYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an indicator dye to each test solution will result in a color change if the test solution contains the macromolecule of interest. The null hypothesis might be that the addition of an indicator dye will not result in a color change in any of the test solutions.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place 16 clean test tubes in a tube rack in four rows of four tubes and label each row A through D and each column 1 - 4.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Label each test tube with its corresponding position in the test tube rack.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">After this, stir solution A thoroughly and use a pipette to add 4 mL of solution A to each tube in Row A.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next, stir solution B until it is well mixed and use a new pipette to add 4 mL of solution B to each tube in Row B.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After stirring solution C thoroughly, add 4 mL of solution C to each tube in Row C.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then add 4 mL of soybean-derived vegetable oil to each tube in Row D.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">When all of the tubes have been filled with their respective test solutions, add 200 mL of water to a 400 mL beaker on a hotplate and set the hotplate to high.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">To perform the Benedict&#39;s Test, add 2 mL of Benedict&#39;s reagent to test tubes A1, B1, C1, and D1 while the water is warming.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Observe the initial color of the tubes.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the water begins to boil, transfer each test tube to the beaker and heat the tubes in the boiling water bath for 3 - 5 minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, record the final color of each of the tubes in the appropriate cells of the table and turn off the hotplate. <a href="https://www.jove.com/CDNSource/lm/labs/22/Table_1_Macromolecules.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To perform an Iodine-Potassium-Iodide Test, first observe the color of the solutions in the test tubes in column 2.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then, add 1 mL of iodine potassium iodide reagent to tubes A2 to D2 and record the final color change in each of the tubes.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For the Sudan IV Test, observe and record the color of the solutions in the test tubes in column 3.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add 2 - 3 drops of Sudan IV reagent to test tubes A3 through D3 and record the color change, if any, in each tube.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">For the Biuret Test, observe the color of the solutions in the test tubes in column 4.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, add 1 mL of Biuret reagent to test tubes A4 through D4 and record the final color in each of the tubes.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Using the results from the indicator tests, each of the four original solutions can be identified as a sugar, starch, lipid, or protein. Refer to the Concepts video to help you determine which tubes contain which macromolecules.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">First, look at the results from the Benedict&#39;s Test. Determine which of the test solutions contain sugar based on your results.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis and the surface area in cm indicated on the y-axis.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, look at the results from the Iodine-Potassium-Iodide Test. Determine which of the test solutions contain starch.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, review the results from the Sudan IV Test. Using these observations, determine if any of the test solutions contain lipids.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Finally, look at the results from the Biuret Test. Determine if any of the test solutions contain proteins.
</div></li></ul></li></ol>

Macromolecules: Identification Using Indicator Solutions - Procedure

Identifying Macromolecules with Indicator Solutions
ExpandHYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an ...