Name: a small volume procedure for viral concentration from water
Uploaded: 2015-02-03T13:00:00.000+00:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

<p class='jove_content'>We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.</p>

<p class="jove_title"> 1. Preparación de Cristalería y alojamientos de los filtros </p><ol style="margin-left: 40px;"><li> A menos que se indique lo contrario, esterilizar todos los artículos de vidrio, soportes y soluciones de filtrado a 121 ° C durante 15 minutos. Para asegurar la esterilidad, cubrir todas las aberturas o las superficies expuestas, ya sea con papel de aluminio o papel cinta asegurado antes de la esterilización. </li><li> Montar aparato de filtración uniendo carcasa del filtro (47 mm de diámetro) a 1 L matraz Erlenmeyer de brazo lateral. Recoger filtros necesarios: filtros de disco electropositivos / electronegativo 47 mm de diámetro. </li><li> Preparar 1 L de extracto de carne 1,5% (floculación, produce partículas de flóculo cuando el pH de la solución se redujo a 3,5, o no floculante, lo que no hace agregada a pH 3,5), con 0,05 M de glicina, disolviendo 15 g de extracto de carne de vacuno en 1 L de agua desionizada y la adición de 3,75 g de glicina. <br /> NOTA: extractos de carne no floculantes requerirán la adición de celita antes de la concentración terciaria. </li><li> Añadir elución polyph sodio aditivoosphate al extracto de carne de vacuno en una concentración de 0,1%. </li><li> Añadir una solución de ácido clorhídrico 1 M, gota a gota, a la mezcla de extracto de carne, solución de pH a pH deseado. Extracto de carne Autoclave durante 15-30 minutos a 121 ° C. </li><li> Mide 0,1 g de fino, medio, o celita partícula grande (para usar con sólo el extracto de carne no floculantes). </li><li> Preparar vacío / aspiración uniendo tubos compatible con erlenmeyer brazo lateral ya la toma de vacío. </li></ol><p class="jove_title"> 2. Preparación de Soluciones y virus Stock </p><ol style="margin-left: 40px;"><li> Preparar 1 L de HCl 1 M. </li><li> Preparar 1 l de 1 M NaOH. </li><li> Prepare la solución de PBS 1x (NaCl 137 mM, KCl 2,7 mM, 1,47 mM K <sub>2</sub> PO <sub>4</sub> y 4,3 mM NaH <sub>2</sub> PO <sub>4).</sub> <ol><li> Ajustar el pH de 1x PBS a 9,0 utilizando 1 M NaOH. </li></ol></li><li> Preparar y diluir de virus a 10 <sup>4</sup> -10 <sup>5</sup> número más probable (NMP) ml <sup>-1</sup> mezclando 1 ml de virus de reserva con 9 ml de PBS (pH7), como se describió previamente <sup>8,13.</sup> Conservar a -80 ° C en alícuotas de 1 ml. <ol><li> Esterilizar virus de stock de filtrado jeringa (0,22 m de tamaño de poro) para eliminar los contaminantes potenciales. </li></ol></li><li> Sin cloro del agua corriente <ol><li> Medida 1 L de agua del grifo estéril usando un cilindro graduado estéril y verter en un vaso de precipitados de 2 l que contenía una barra de agitación magnética. </li><li> Añadir 0,7 g de tiosulfato de sodio y mezclar hasta que se disuelven los gránulos. Ajustar el pH del agua del grifo para 7-7,5 si es necesario el uso de HCl 1 M o solución de NaOH 1M. </li><li> Pico virus Deshielo (1 ml) y se añade todo el volumen de pico a 1 l de agua del grifo estéril sin cloro. Mezclar durante 10 min. </li><li> Preparar un blanco método (el agua del grifo sin cloro y sin adenovirus) con cada experimento y el proceso de la misma manera que la muestra de púas. </li></ol></li></ol><p class="jove_title"> 3. Preparación de Millipore Filtro Aparato </p><ol style="margin-left: 40px;"><li> Retirar la cobertura estéril sobre la carcasa del filtro. Asegurarajuste adecuado entre la carcasa del filtro tazón superior y pantalla de filtro inferior para evitar fugas de la muestra durante la filtración. </li><li> Retirar la cobertura estéril de 1 frasco L Erlenmeyer. Asegúrese de que el corcho de la unidad de filtro se ajusta perfectamente en la abertura superior de erlenmeyer, creando un buen sello. </li><li> Retire el tazón superior de la unidad de filtro de malla del filtro inferior y colocar 47 mm disco de filtro electropositivo de lleno sobre la pantalla con pinzas estériles. Adjuntar filtro tazón vivienda a la pantalla inferior y ajustar en su lugar girando en sentido antihorario. </li></ol><p class="jove_title"> 4. Pulse de filtración de agua (Concentración Primaria) </p><ol style="margin-left: 40px;"><li> Medir 100 ml de virus se dispararon el agua del grifo usando estéril probeta. </li><li> Vierta 100 ml de virus se dispararon el agua del grifo en la carcasa del filtro que contiene o bien un filtro de 47 mm de vidrio plisado / celulosa o un filtro de disco de fibra nano-alúmina / vidrio. </li><li> Deje que el agua pase lentamente a través del filtro. Aplicar vacío suave para eliminar los residuos restantes oagua del grifo f de la superficie del filtro. </li><li> Si se utiliza el tratamiento con tripsina, añadir 5 ml de tripsina al 0,25% para filtrar superficie e incubar durante 5 minutos. </li><li> Lavar la tripsina de la superficie del filtro utilizando 50 ml de agua desionizada estéril. </li></ol><p class="jove_title"> 5. La elución Virus del filtro </p><ol style="margin-left: 40px;"><li> Retire la carcasa del filtro de 2 L frasco Erlenmeyer, y el lugar en frasco de 250 ml brazo lateral. </li><li> Medir 100 ml de extracto de carne utilizando cilindro graduado. Vierta lentamente 100 ml de extracto de carne en caja del filtro. </li><li> Permitir extracto de carne para verter a través del filtro, capturando extracto de carne en una pequeña (250 ml) matraz Erlenmeyer. Aplicar vacío para tirar a través de extracto de carne residual del filtro. </li></ol><p class="jove_title"> 6. Celite Concentración Secundaria </p><ol style="margin-left: 40px;"><li> Frascos de transferencia que contiene los virus eluidas a una placa de agitación. Añadir estéril barra de agitación magnética y colocar en placa de agitación, mezcla para crear ligero vórtice. </li><li> Añadir 0,1 g de celitepolvo a 100 ml de extracto de carne y permitir de celite para dispersar. Coloque la sonda de pH estéril en extracto de carne. </li><li> Añadir gota a gota HCl 1 M de extracto de carne para alcanzar un pH de 4,0. Permita mezclar lentamente durante 10 min. </li><li> Coloque 47 mm pre-filtro en la carcasa del filtro (como se describió anteriormente) usando un matraz Erlenmeyer de 2 l. </li><li> Verter 100 ml de extracto de carne de vacuno / de la mezcla de celite sobre pre-filtro. Permita que el extracto de carne vierta a través. Aplicar ligero vacío para salir adelante extracto de carne residual del filtro. </li><li> Coloque el metal clip de tubo en el extremo del tubo de salida del filtro. Adjuntar estéril 15 ml tubo de recogida de polipropileno para clip de tubo de metal. Coloque la carcasa del filtro de nuevo en erlenmeyer. </li><li> Añadir 5 ml de tampón 1x PBS, pH 9,0 sobre celite recogida en pre-filtro. </li><li> Permita PBS gotee a través de pre-filtro. Aplicar ligero vacío para salir adelante PBS residual en 15 ml tubo de recogida. </li></ol><p class="jove_title"> 7. Orgánica floculación Concentración Secundaria </p><ol style="margin-left: 40px;"><li>La transferencia de los vasos que contienen el virus se eluyó a una placa de agitación. Añadir una barra de agitación magnética estéril a 100 ml de extracto de carne y mezclar para crear ligero vórtice. </li><li> Coloque la sonda de pH estéril en extracto de carne. </li><li> Añadir 1 M de HCl gota a gota a extracto de carne y ajustar el pH a 3,5. Mezclar durante 30 min. </li><li> Verter el eluato en 250 ml tubos cónicos de centrífuga y centrifugar a 2500 xg durante 15 min a 4 ºC. </li><li> Verter el sobrenadante con cuidado de no de no perturbar el sedimento. Resuspender el precipitado en 5 ml de 1x PBS, pH 9. </li><li> Centrifugar la suspensión a 4.000 xg durante 10 min a 4 ºC. Verter el sobrenadante y desechar el sedimento. </li><li> Ajustar el pH a 7-7,5, esterilizar por filtración a través de filtro de jeringa de 0,22 micras y congelación a -80 ° C. </li></ol><p class="jove_title"> 8. Viral Nucleic Acid Extracción </p><ol style="margin-left: 40px;"><li> Extraer ácidos nucleicos virales utilizando un kit comercial apropiado de acuerdo con las instrucciones del fabricante. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 9. AdV 40/41 qPCR </p><ol style="margin-left: 40px;"><li> Utilice cebadores y sondas, así como la mezcla de reacción y perfiles de ciclo térmico según lo publicado <sup>8,13.</sup> <br /> NOTA: límite estimado de detección del ensayo es de aproximadamente 5 unidades qPCR por reacción. </li><li> Añadir componentes de las mezclas de amplificación de PCR en concentraciones como sigue: Tris 10 mM (pH 8,3), KCl 50 mM, 4,5 mM de MgCl <sub>2,</sub> mM dNTPs 10, 10 mM cebadores y sonda 1 M y 0,5 l de polimerasa de manera que los volúmenes finales de 45 l se puede añadir al número apropiado de pozos dentro de una placa de 96 pocillos de reacción PCR. </li><li> Cargar 45 l de mezcla de amplificación PCR a cada pocillo apropiado de una de 96 pocillos, la placa de PCR reacción rápida. </li><li> Añadir 5 l de ADN extraído de la muestra a los pocillos apropiados de la placa de PCR. </li><li> Cubra la placa con junta de la tapa resistente al calor, girar la placa para mover cualquier gota a fondo de pozos de la placa de PCR. </li><li> Establecer ciclos de amplificación por PCR como follOWS: 95 ° C durante 10 min, seguido por 40 ciclos de 95 ° C durante 15 segundos y 60 ° C durante 1 min. </li></ol><p class="jove_title"> 10. Análisis de datos </p><ol style="margin-left: 40px;"><li> Analizar mediciones por triplicado de las diluciones en serie de 5 veces de las muestras que abarcan 1: 5 a 1: 625 dilución gama con el fin de determinar el número más probable (MPN) de las partículas virales, como se describió previamente <sup>8,13.</sup> </li><li> Analizar diluciones en serie de 5 veces de los picos experimentales de adenovirus que abarcan 1: 5 a 1: 15.625 rango de dilución para determinar el número más probable (MPN) de las partículas virales. </li><li> El uso de la calculadora EPA MPN (http://www.epa.gov/nerlcwww/online.html), ajustar la calculadora para tener en cuenta el tipo de dilución (1: 5 ó 1:10), el número de diluciones realizado, y el número de replicar muestras en serie de dilución. </li><li> Realizar log <sub>10</sub> transformación de los datos, seguido por la normalización a una unidad de volumen <em>(por ejemplo,</em> 1 ml, 100 ml, <em>etc.</em> </em&gt;). Calcular recuperaciones para experimento dado dividiendo la cantidad de partículas virales presentes en la muestra se concentra con el número de partículas virales presentes en los picos experimentales. </li><li> Evaluar el efecto de las diferentes variables experimentales <em>(por ejemplo,</em> diferentes tipos de celite, se rangos de pH, diferentes tipos de filtros y diferentes técnicas de concentración secundarios) sobre recuperaciones AdV mediante la realización de pruebas estadísticas paramétricas o no paramétricas <em>(por ejemplo,</em> prueba t pareada, uno o dos -Way ANOVA) dependiendo de la distribución de los datos y el diseño experimental. </li></ol>

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J., De Leon, R., Sobsey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+purification+of+beef+extract+mock+eluates+from+water+samples+for+the+detection+of+enteroviruses,+hepatitis+A+virus,+and+Norwalk+virus+by+reverse+transcription-PCR.">Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and Norwalk virus by reverse transcription-PCR.</a> <em style='font-style: italic'>Applied and Environmental Microbiology</em>. <b>61</b>, 531-537 (1995).</li></ol>

<p class='jove_content'>No competing financial interests. </p>
<p class='jove_content'>The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.</p>

<p class="jove_content"> Filtros electropositivos son útiles en la concentración de virus del agua; sin embargo, estos filtros pueden diferir en su estructura y composición, que a su vez podría alterar su eficacia. Para agravar este problema, las estructuras de la cápside y cargos varían entre las cepas de virus que requieren ser adaptados técnicas de concentración para asegurar una recuperación óptima <sup>15.</sup> A través de simples modificaciones de las técnicas de concentración existentes <em>(por ejemplo,</em> los filtros el...

<p class="jove_content"> Virus entéricos humanos son agentes causantes importantes de enfermedades de origen hídrico <sup>1-3,</sup> pero generalmente están presentes en números bajos en aguas ambientales contaminadas, por lo que su detección difícil sin concentración. Los procedimientos utilizados para concentrar virus incluyen típicamente una etapa de filtración, seguido de elución de filtro, y la concentración secundaria del eluato filtro. Un procedimiento de filtración común se basa en la utilización de membranas cargadas tales como filtros electropositivos (recientemente revisado en <sup>4,5).</sup> Estos filtros se basan en la captura de los virus en suspensión en el agua utilizando interacciones electrostáticas entre la superficie del filtro (con carga positiva) y partículas de virus específicos (con carga negativa). Dos filtros electropositivos que están disponibles comercialmente se basan en esta tecnología, los filtros de vidrio / celulosa y fibra nano-alúmina / vidrio. Los costes de filtro de vidrio / celulosa son hasta 10 veces mayor que la de la fibra nano-alúmina / vidrio, que limitan el uso del vidrio / cefiltros llulose para la vigilancia del virus de rutina. Estudios recientes han concluido diferencias son nominales entre estos dos filtros en la recuperación de los enterovirus desde <sup>6,7</sup> del agua ambiente, lo que justifica el uso de una alternativa de filtro más barato. Otras opciones de filtro, como los filtros electronegativos y de lana de vidrio se han estudiado, sin embargo, o bien requieren el tratamiento previo de las fuentes de agua (filtros electronegativos) o no están disponibles en el mercado (filtros de lana de vidrio). El desarrollo de procedimientos de concentración de virus se ha centrado principalmente en la optimización de las técnicas de concentración primaria (filtros) con el fin de mejorar la recuperación de virus de agua. Sin embargo, los procedimientos de concentración secundarios, que reducen el volumen de eluyente típicamente de 1 L a mililitro volúmenes, también pueden tener un impacto significativo en las recuperaciones de virus <sup>8.</sup> </p><p class="jove_content"> Concentración secundaria de virus entéricos típicamente se basa en un agente floculante, tales como algunos tipos de extracto de carne (floccu orgánicamento) o floculación de la leche desnatada <sup>9-12</sup> para eliminar las partículas de virus de las superficies del filtro. Recientemente, otro procedimiento de concentración secundaria utilizando extracto de carne junto con la adición de celita (partículas finas) ha mostrado ser prometedor para la recuperación de adenovirus, enterovirus y norovirus <sup>8,13,14.</sup> Obras de concentración Celite bajo principios similares a la del método de floculación orgánica en que las partículas de virus se unen a y se liberan de partículas (floc o de celite) alterando el pH de la solución en suspensión. Las comparaciones entre estas dos técnicas de concentración secundarias han sido evaluadas en la recuperación de adenovirus (ADV) de pinchos tipos 40 y 41 <sup>8.</sup> Este estudio concluyó que las dos técnicas de concentración secundarias fueron estadísticamente similares en la recuperación de adenovirus. Sin embargo, el método de floculación orgánica requiere una 30 min. de incubación a pH 3,5, mientras que la técnica de celite requiere una incubación más corto (10 min) a pH 4,0. El flocculat orgánicaion también requiere el uso de equipo de laboratorio caro (centrifugadoras) para recoger las partículas de flóculos durante la concentración terciaria, la técnica de celite en contraste utiliza sólo el equipo de laboratorio básico (filtración a vacío) para separar partículas de Celite de la suspensión. </p><p class="jove_content"> Ciertas combinaciones de filtros y técnicas de elución secundarias también pueden afectar a las recuperaciones de virus. Un estudio concluyó que ciertas combinaciones de (filtros electropositivos) primarias y secundarias técnicas de concentración (Celite o floculación orgánica) tuvieron un impacto significativo de la recuperación del adenovirus <sup>13.</sup> Estos hallazgos sugieren que se requiere la optimización a fin de recuperar de manera óptima virus diana a partir de una matriz de agua dado al utilizar estas técnicas. La optimización es una pérdida de tiempo y arduo proceso de muchos investigadores evitan activamente desde numerosas variables serán evaluados (tipo de filtro / marca, solución de elución de pH, celita / floculación orgánica). </p><p class="jove_content"> Para este study, se desarrolló un procedimiento para identificar las condiciones óptimas para la concentración de virus a partir de agua usando cepas de adenovirus humano de púas 40 y 41. Presumiblemente, ya que cada tipo virus muestra una morfología de la cápside único y carga específica de la cápside, pueden necesitar ser optimizado para cada virus protocolos de concentración apuntar a fin de alcanzar la recuperación viral óptima. Este estudio proporciona un enfoque para AdV 40 y 41 de concentración por: 1) la evaluación de la recuperación de virus en el agua del grifo, usando discos de filtro electropositivos seguido por 2) evaluación de un método de floculación orgánica establecido frente a la técnica de celite como una concentración secundaria, y 3) evaluación de tampones de elución para la concentración terciaria. </p>

<table><tbody><tr><td>Adenovirus 40 stock</td><td>ATCC</td><td>VR-931</td><td></td></tr><tr><td>Adenovirus 41 stock</td><td>ATCC</td><td>VR-930</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>Fluka Chemical Co.</td><td>72051</td><td></td></tr><tr><td>Celites #577</td><td>Fluka Chemical Co.</td><td>22142</td><td></td></tr><tr><td>NanoCeram 47 mm</td><td>Argonide</td><td>N/A</td><td></td></tr><tr><td>1MDS 47 mm</td><td>3M</td><td>6408502</td><td></td></tr><tr><td>AP-20 Prefilter 47 mm</td><td>Millipore Corp.</td><td>AP2004700</td><td></td></tr><tr><td>Glycine&amp;nbsp;</td><td>Sigma</td><td>50046-1KG</td><td></td></tr><tr><td>Sodium Polyphosphate</td><td>Acros Organics</td><td>390930010</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>P5368</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher</td><td>A481-212</td><td></td></tr><tr><td>BBL Beef Extract</td><td>BD Biosciences</td><td>212303</td><td></td></tr><tr><td>Difco Beef Extract</td><td>BD Biosciences</td><td>211520</td><td></td></tr><tr><td>ABI 7900 Real-time PCR system</td><td>ABI</td><td>N/A</td><td></td></tr><tr><td>Stainless Steel Filter Housing</td><td>Millipore Corp.</td><td>XX2004720</td><td></td></tr><tr><td>Blood DNA Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>51104</td><td></td></tr><tr><td>EPA MPN Calculator</td><td></td><td></td><td>http://www.epa.gov/nerlcwww/online.html</td></tr></tbody></table>

a small volume procedure for viral concentration from water

<p class='jove_content'>Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract. </p>

<html><p class="jove_content"> Selección de Celite </p><p class="jove_content"> Tres tipos diferentes de celita fueron probados antes de la selección de la mejor variante de realización. Celites con bien a las partículas de tamaño mediano producen los más altos recuperaciones de adenovirus. El uso de celites más grandes resultó en menores recuperaciones tanto para AdV40 y 41 (rango de 32% -100%) <strong>(Figura 1).</strong> Promedio de recuperaciones de adenovirus 40 eran 144% ± 52% (muy bien), 115% ± 28% (medio) y 82% ± 53% celites ...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

<p class='jove_content'>An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. </p>

Un procedimiento de volumen pequeño para la concentración viral del Agua

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

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10.0K vistas.  U.S. EPA. An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. 

Video: A Small Volume Procedure for Viral Concentration from Water

Steam 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="">Steam Distillation<button class="expand">Expand</button>
	<p>In this experiment, you will perform a steam distillation to extract an essential oil from an orange peel.</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 performed in a hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">First, you will need to assemble the steam distillation apparatus. Check the apparatus set up by your instructor.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Set the two stands next to each other in the hood. Place the stir plate on the base of one of the stands and set the heating mantle on top of it. Plug the heating mantle into the temperature controller, but do not turn it on yet.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Now, obtain a watch glass with 3 dried orange peels from your instructor. Weigh the orange peels on the balance and record the mass of the orange peels in your notebook.
		<h4><strong>Table 1: Mass of essential oil recovered</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" height="166" width="476">
			<tbody>
				<tr>
					<td>Temperature of steam distillation (&#176;C)</td>
					<td></td>
				</tr>
				<tr>
					<td>Mass of the orange peels (g)</td>
					<td>&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
				</tr>
				<tr>
					<td>Mass of empty flask (g)</td>
					<td></td>
				</tr>
				<tr>
					<td>Mass of flask + essential oils (g)</td>
					<td></td>
				</tr>
				<tr>
					<td>Mass of essential oils (g)</td>
					<td></td>
				</tr>
				<tr>
					<td>Percent mass of essential oil recovered</td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/61/Table_1._Mass_of_essential_oil_recovered.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Bring your dried peels, funnel, spatula, and the 500-mL round-bottom flask to the instructor&#39;s bench and place the peels in the blender. Add about 4 mL of deionized water to the peels. Then, blend them until they are well mixed and smooth.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Using the funnel, transfer the blended peels to the round-bottom flask. Clamp the flask to the stand above the heating mantle.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure 200 mL of deionized water using your graduated cylinder and transfer it to the round-bottom flask using a funnel. Remove the funnel and add a stir bar to the flask.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Locate the Claisen adapter and apply a thin layer of grease to the joint. Then, insert the Claisen adapter into the flask.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Grease the thermometer adapter and insert it into the distilling head.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Gently insert the thermometer into the distilling head by pushing and twisting through the adapter until the thermometer bulb is below the bend of the distilling head.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Lightly grease the lower joint of the distilling head, and then insert it into the outer arm of the Claisen adapter.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Now, obtain the condenser and carefully clamp it to the second stand. It should be oriented nearly horizontally, with the end closest to the distilling head slightly higher.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Lightly grease the condenser and the side joint of the distilling head before connecting them.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Next, attach one piece of rubber tubing to the inlet of the condenser, which is the port furthest away from the distilling head. Attach the other end to the tap water faucet in the hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Connect the second piece of rubber tubing to the other port, which is the outlet, and place the end of the tubing in the drain.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Locate the separatory funnel and make sure that the stopcock is closed by turning it until it is fully horizontal.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Insert the separatory funnel into the Claisen adapter, and add approximately 100 mL of deionized water to the funnel.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Pick up the connecting joint and attach it to the end of the condenser.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Then, attach a 250-mL round-bottom flask to the connecting joint using a plastic clip.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Finally, check all of the joints to make sure they are tightly connected and then secure each one with a plastic clip.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Now that the steam distillation apparatus is fully assembled, start the procedure. First, slowly turn the tap water on to fill the condenser with water. <strong>Note</strong>: Be sure to turn the water on slowly, or the tubing may pop off and cause a flood.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Next, turn on the magnetic stirrer at the lowest setting and set the temperature controller to the appropriate setting to reach about 90 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Once bubbles start forming in the mixture and liquid is dripping into the collection flask, adjust the heat to increase or decrease the rate of distillation to about 20 drops/min.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Record the temperature on the thermometer in your notebook. Now, carefully open the stopcock on the separatory funnel to slowly add water to the flask. Your goal is to add water at the same rate as distillation, about 20 drops/min.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Allow the distillation to proceed and collect about 50 mL of distillate.<strong> Note:</strong> Be patient, since this should take about 2 hours. The liquid in the flask will become less cloudy as more oil is collected.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Once you have collected enough distillate, turn off the heat. Then, shut off the water to the condenser and carefully raise the entire setup.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Allow it to cool completely before removing the heating mantle and disassembling the glassware.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Empty the separatory funnel of any remaining water and pour the orange pulp down the drain.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Extraction of Essential Oil<button class="expand">Expand</button>
	<p>Liquid-liquid extraction is performed in a separatory funnel and is used to move a solute to another liquid in which it is more soluble. The two liquids must be immiscible and are usually an aqueous solution and a non-water-soluble organic liquid. Upon mixing, the solute is extracted into the other phase. Once the liquids settle into layers, they can be drained from the funnel one at a time.</p>
	<p>In the next part of the experiment, liquid-liquid extraction will be used to extract the essential oil from the water into an organic solvent. You&#39;ll then evaporate the organic solvent to recover the essential oil.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Before starting the experiment, first, make sure that the separatory funnel is rinsed and clean.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Label the two 250-mL beakers as &#8216;organic layer&#8217; and &#8216;aqueous layer&#8217;. Then, attach a ring support to one of the stands.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Place the separatory funnel in the support. Make sure that the stopcock is closed and the separatory funnel is secure. Then, transfer the distillate into the separatory funnel.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Measure 20 mL of diethyl ether at the instructor&#39;s hood and bring it back to your workstation. Pour the diethyl ether into the separatory funnel with the distillate. <strong>Note:</strong> You should see the immiscible liquids form two layers in the funnel. The top layer is the diethyl ether.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Apply a thin layer of vacuum grease to the separatory funnel stopper and insert it into the top of the funnel.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Hold the stopper in place and shake the separatory funnel vigorously for about 1 min.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Occasionally, vent the funnel by turning it upside down and opening, then closing the stopcock. Be sure to do this inside of the hood and away from your face.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Once you have finished mixing the contents, vent the funnel again, close the stopcock, and then place the funnel in the ring attached to the stand.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Allow the aqueous and organic layers to fully separate. Then, place the aqueous layer beaker under the separatory funnel.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Remove the stopper and slowly open the stopcock to drain the aqueous layer into the beaker. Carefully watch the solution drain and be sure to close the stopcock before the next layer is drained.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Replace the aqueous layer beaker with the organic layer beaker. Open the stopcock and drain the organic layer into its beaker.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Now, close the stopcock and return the contents of the aqueous layer beaker to the separatory funnel, add 20 mL of diethyl ether, and repeat the extraction.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Open the stopcock and drain the aqueous layer into the appropriate beaker, and then drain the organic layer into the organic layer beaker, adding it to the organic layer from the first extraction.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Now, obtain magnesium sulfate in a weighing boat and scoop a small spatula of it into the organic layer beaker. Swirl the beaker, and then let it sit for about 20 min.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">After 20 min, remove the separatory funnel and ring and mount a regular clamp to the lab stand.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Weigh an empty 125-mL Erlenmeyer flask and record the mass in your notebook. Then, clamp the flask to the stand and place a conical glass funnel in its neck.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Fold your filter paper in half and in half again. Then, open the filter paper on one side to create a cone. Place the cone inside the conical funnel.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Now, filter the magnesium sulfate from the organic layer.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Next, set your hot plate to 40 &#176;C and place a large beaker containing 400 mL of water on it. Place the Erlenmeyer flask in the warm water bath and allow the organic solvent to evaporate in the hood.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Once the organic solvent has fully evaporated, remove the Erlenmeyer flask from the water bath and dry the outside with paper towels.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Weigh the cooled flask and record the mass in your notebook.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Now that the experiment is complete, be sure to properly clean up your workspace by disposing of all solutions in the non-halogenated waste.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Wash all of the glassware using detergent and then rinse them with acetone and deionized water.</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">Determine the mass of the oil extracted from the orange peel by subtracting the mass of the empty flask from the mass of the flask with oil.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Calculate the percentage of oil in the orange peel using the original mass of the dried peel. Typically, the steam distillation method recovers about 0.9 &#8211; 4 wt% of an orange peel&#8217;s mass as essential oil.</div>
		</li>
	</ul>
	<p>You distilled the essential oil in this lab at close to 100 &#176;C. The orange oil consists mainly of (+)limonene, which has a boiling point of 176 &#176;C. Thus, the steam distillation technique lets us extract it from the orange peel at a temperature that will not compromise its structure.</p>
	</li>
</ol>

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

Destilación al vapor

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

	Steam DistillationExpand
	In this experiment, you will ...

Educación

JoVE Lab Manual

Química

Lab Manual Sub Articles

Extraction

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

Extracción

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

Destilación simple

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

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

Scientific Measurement and Lab Skills

Two-Dimensional Graphs, Curve Fitting, and Standard Deviation - Procedure

Medición Científica y Habilidades de Laboratorio

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

	Measuring Diameter and CircumferenceExpand
	Error can be introduced in any experiment, even ...

Stoichiometry, Product Yield, and Limiting Reactants

Estequiometría, rendimiento del producto y reactivos limitantes

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

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

Acid and Base Concentrations

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of ~0.1 M NaOH<button class="expand">Expand</button>
	<p>In the first part of the lab, you will use a 50% w/w solution of NaOH to prepare 500 mL of ~0.1 M. The 50% w/w NaOH is indicative of its weight ratio. For example, if the instructor prepared 150 mL of the 50% w/w NaOH solution, then 150 g of NaOH was dissolved in 150 g of water, and the total weight of the solution is 300 g.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">To begin, put on the appropriate personal protective equipment, including gloves, chemical splash goggles, and a lab coat.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Calculate the molarity of 50% w/w NaOH, which is M<sub>1</sub> in the dilution formula.<strong> Note: </strong>The density of 50% w/w NaOH is 1.53 g/mL, and the molar mass of NaOH is 39.998 g/mole.
		<h4>Table 1. Preparation of 0.1 M NaOH from 50% w/w NaOH</h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td>Density of 50% w/w stock solution</td>
					<td style="text-align: center;">1.53 g/mL</td>
				</tr>
				<tr>
					<td>Molar mass<sub>NaOH</sub></td>
					<td style="text-align: center;">39.998 g/mol</td>
				</tr>
				<tr>
					<td>Mass of NaOH in 50% w/w stock solution (mg)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Total mass of 50% w/w stock solution</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of 50% w/w stock solution (mL)</td>
					<td></td>
				</tr>
				<tr>
					<td>Moles of NaOH in 50% w/w solution (mol)</td>
					<td></td>
				</tr>
				<tr>
					<td>Molarity of 50% w/w stock solution (M<sub>1</sub>)</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume of 50% w/w solution needed (V<sub>1</sub>)</td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/44/Table_1._Preparation_of_0.1_M_NaOH.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Use the dilution formula to determine the volume of 50% w/w NaOH needed to prepare 500 mL of ~0.1 M NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label the 500 mL polyethylene bottle as &#8216;~0.1 M NaOH&#8217;.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Adjust the volume on a 1-mL pipette to the value calculated and use it to transfer the 50% w/w NaOH to the polyethylene bottle.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use a 100-mL graduated cylinder to measure the amount of water and pour it into the bottle containing the NaOH. Cap the bottle and invert it several times to mix the solution. <strong>Note:</strong> You will need 500 mL minus the calculated volume of NaOH.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Standardization of 0.1 M NaOH<button class="expand">Expand</button>
	<p>After you have prepared 0.1 M NaOH, determine its exact concentration, or standardize it, using the acid-base titration method. In this technique, a base like NaOH is slowly added to an acid like potassium hydrogen phthalate (KHP). The chemical reaction that takes place in the flask is a neutralization reaction. In this neutralization reaction, one mole of base neutralizes one mole of acid, resulting in salt and water. This reaction is performed in the presence of the indicator phenolphthalein, which is colorless at the start of the reaction when the pH is acidic. The indicator turns pink as soon as enough NaOH is added to the flask to make the pH basic.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">Label the three Erlenmeyer flasks as &#8216;A&#8217;, &#8216;B&#8217;, and &#8216;C&#8217;.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Weigh 0.5 &#8211; 0.7 g of KHP for each of the flasks and record the mass for each. <strong>Note: </strong>Try to measure the same mass of KHP for all three flasks.
		<h4><strong>Table 2. Standardization of NaOH</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Molar mass<sub>KHP</sub> = 204.23 g/mol </strong></td>
					<td style="text-align: center;"><strong>Flask A</strong></td>
					<td style="text-align: center;"><strong>Flask B</strong></td>
					<td style="text-align: center;"><strong>Flask C</strong></td>
				</tr>
				<tr>
					<td>Mass of KHP (g)</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Volume<sub>initial NaOH</sub>(mL)</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Volume<sub>final NaOH</sub> (mL)</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Volume<sub>NaOH</sub> (mL)</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Moles of KHP</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Moles of NaOH</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Molarity of NaOH</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Average molarity</td>
					<td></td>
				</tr>
				<tr>
					<td>Standard deviation</td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/44/Table_2._Standardization_of_NaOH.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Add 50 mL of deionized water to each flask, and use a glass stirring rod to stir the solutions until they appear homogeneous.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Add 2-3 drops of phenolphthalein to each of the three flasks.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Set up the titration apparatus. Attach the burette clamp to the ring stand, and securely clamp the 50-mL glass burette to it. Make sure the valve of the burette is closed.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Label a 400-mL beaker as &#8216;waste&#8217; and place it under the burette. Rinse the burette by pouring about 5 mL of 0.1 M NaOH into the burette. Open the burette valve to allow the NaOH to flow into the beaker.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Close the valve and fill the burette with slightly more than 50 mL of NaOH. Release any air bubbles present in the tip of the burette by opening and closing the burette valve. Record the starting volume of NaOH.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place flask A below the tip of the burette, and titrate the solution using 1 mL volumes of NaOH. Swirl the solution after each addition. Continue adding 1 mL volumes to the flask until the pink color persists. This is considered the endpoint. Record the volume of 0.1 M NaOH added to achieve the endpoint.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Repeat the titration for flasks B and C. This data will be used to calculate the actual concentration of NaOH.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Titration of phosphoric acid<button class="expand">Expand</button>
	<p>In this experiment, we will determine two of the three pKa values for the triprotic acid, phosphoric acid, using acid-base titration. In this neutralization reaction, phosphoric acid reacts with NaOH to form water and the salt, sodium phosphate.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Attach the drop counter to the ring stand, below the burette clamp. Secure the plastic burette so that its tip is just above the drop counter.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Connect the drop counter to the data acquisition system, and make sure the two valves of the plastic burette are both in the closed position.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Place the waste container below the burette and pour a few mL of 0.1 M NaOH into the burette. Open both valves to drain the NaOH into the waste beaker. Then close the valves.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Fill the plastic burette with 25 mL of 0.1 M NaOH. Drain about 5 mL into the waste beaker&#8212;enough so that NaOH fills the burette tip. Make sure there are no air bubbles, then close the valves.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Calibrate the drop counter. Replace the waste beaker beneath the burette with a 10-mL graduated cylinder. Then, open the bottom valve on the burette, while keeping the top valve closed. Turn on the data acquisition system and set it to &#8216;drop counting mode&#8217;.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Slowly open the top valve to very slowly release drops, ideally at one drop every 2 s. Allow the drops to empty from the burette until there is 9-10 mL of 0.1 M NaOH in the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Close the bottom valve and leave the top valve as is. Read the volume of NaOH in the graduated cylinder to the first decimal place and enter this value in the data acquisition system. Record the value for &#8216;drops/mL&#8217;. Then, discard the NaOH in the graduated cylinder into the aqueous waste beaker.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Calibrate the pH sensor before starting the titration. Connect the pH sensor to the data acquisition system, then select &#39;Calibrate&#39;. Rinse the bulb of the pH sensor with deionized water before inserting it into the pH 7 buffer. Leave the sensor submerged until the voltage stabilizes, then accept the measurement.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">Rinse the bulb with deionized water and insert it into the pH 10 buffer. Allow the voltage to stabilize, then accept the measurement.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Rinse the bulb of the pH sensor again and slide it through the designated slot in the drop counter.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Measure 40 mL of deionized water into a clean 100-mL beaker, then, transfer 1 mL of phosphoric acid into the beaker of water.</div>
		</li>
		<li data-sub-note-item="3-13">
		<div class="lab-step">Place the beaker on the stir plate under the drop counter. Carefully slide the pH sensor into the beaker.</div>
		</li>
		<li data-sub-note-item="3-14">
		<div class="lab-step">Add a stir bar to the beaker, and turn the stir setting onto high. Start collecting data on the acquisition device. Then, open the bottom valve on the burette. <strong>Note: </strong>The drop rate should be about 1 drop every 2 s. After the first drop is released, check to see data are being recorded.</div>
		</li>
		<li data-sub-note-item="3-15">
		<div class="lab-step">Continue the titration until the pH meter reads pH 12. Then, stop acquiring data and close the valve on the burette. Save your data on a flash drive.</div>
		</li>
		<li data-sub-note-item="3-16">
		<div class="lab-step">To clean up your workspace, check the pH of all waste solutions using pH paper. Neutralize all acidic aqueous waste with baking soda and all basic waste with citric acid. Add enough of either baking soda or citric acid to the solution until it stops bubbling.</div>
		</li>
		<li data-sub-note-item="3-17">
		<div class="lab-step">Flush all neutralized solutions down the sink with copious amounts of water.</div>
		</li>
		<li data-sub-note-item="3-18">
		<div class="lab-step">Wash all glassware.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="4-1">
		<div class="lab-step">In the first part of this lab, you standardized a solution of NaOH using KHP to determine its actual concentration. Now, let&#39;s see how close the standardized concentration is to the 0.1 M concentration that was prepared. Determine the number of moles of KHP that was added to each flask, and by extension, the moles of NaOH. Once the solution is neutralized, the molar quantities of KHP and NaOH are equal.</div>
		</li>
		<li data-sub-note-item="4-2">
		<div class="lab-step">Calculate the molarity of NaOH based on the total volume of NaOH that was added to each flask. The actual concentration is lower than the expected 0.1 molarity. This is because NaOH is hygroscopic, so it is difficult to weigh it accurately.</div>
		</li>
		<li data-sub-note-item="4-3">
		<div class="lab-step">Plot the results for the phosphoric acid titration (pH vs. Volume of NaOH). Phosphoric acid is a weak triprotic acid, meaning that it has the potential to provide three protons per molecule when it dissociates in aqueous solutions. Phosphoric acid has three pKa values, one for when each proton is dissociated.</div>
		</li>
		<li data-sub-note-item="4-4">
		<div class="lab-step">Look at the data. There are two sigmoidal curves, indicating two equivalence points. Each equivalence point corresponds to a dissociation constant, Ka, of phosphoric acid. <strong>Note: </strong>You stopped the experiment once the pH reached 12, so you only measured two of the three Ka values.</div>
		</li>
		<li data-sub-note-item="4-5">
		<div class="lab-step">Plot the first derivative of the titration curve. The equivalence points are represented by the curve maximums.
		<h4><strong>Table 3. Titration of phosphoric acid</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td>Volume<sub>1<sup>st</sup> equivalence point</sub> (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume<sub>1<sup>st</sup> half-equivalence point</sub> (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume<sub>2<sup>nd</sup> equivalence point</sub> (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume<sub>2<sup>nd</sup> half-equivalence point</sub> (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>1<sup>st</sup> pKa<sub>measured</sub></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>1<sup>st</sup> pKa<sub>theoretical</sub></td>
					<td style="text-align: center;">2.16</td>
				</tr>
				<tr>
					<td>2<sup>nd</sup> pKa<sub>measured</sub></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>2<sup>nd</sup> pKa<sub>theoretical</sub></td>
					<td style="text-align: center;">7.21</td>
				</tr>
				<tr>
					<td>Moles of NaOH</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Moles of H<sub>3</sub>PO<sub>4</sub></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Molarity of H<sub>3</sub>PO<sub>4</sub></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/44/Table_3._Titration_of_Phosphoric_Acid.xlsx" target="_blank">Click Here to download Table 3</a></div>
		</li>
		<li data-sub-note-item="4-6">
		<div class="lab-step">Find the first half-equivalence point by taking the volume of NaOH corresponding to the first equivalence point and dividing it by 2. At the half-equivalence point, the concentrations of undissociated acid and its conjugate base are the same, and the pH is equal to the pKa.</div>
		</li>
		<li data-sub-note-item="4-7">
		<div class="lab-step">Look up the pH at this volume from your table of data to get a more precise value. This corresponds to the first pKa, which is reported in the literature as 2.16.</div>
		</li>
		<li data-sub-note-item="4-8">
		<div class="lab-step">Repeat this to find the second pKa. The second half-equivalence point is located midway between the first and second equivalence points, which should give a pKa of about 7.2.</div>
		</li>
	</ul>
	</li>
</ol>

Acid-Base Titration: Calculating pH, Strength, and Concentration - Procedure

Concentraciones de ácido y base

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

	Preparation of ~0.1 M NaOHExpand
	In the first part of the lab, you will use a 50% w/w solution ...

Solubility

Solubility: Solubility Equilibrium and Thermodynamics - Procedure

Solubilidad

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

	Creating a Saturated SolutionExpand
	In this experiment, you will create a saturated solution ...

Concentration Dependence

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Concentration Dependence<button class="expand">Expand</button>
	<p>In this experiment, you will combine varying concentrations of aqueous HCl and sodium thiosulfate to make solid sulfur, which rapidly gathers into opaque yellow particles at a certain sulfur concentration. Since the reaction solution starts clear and colorless, you can easily tell when it reaches that concentration.</p>
	<p>You&#39;ll use the same test tube and total volume each time, so it will take the same amount of sulfur to make each solution completely opaque. Thus, you&#39;ll measure the reaction progress by timing how long that takes. You&#39;ll then use that data to estimate the reaction orders of the individual reactants and of the overall reaction.</p>
	<p>Before starting the lab, make a table listing the reactant concentrations, the time to solution opacity, and the solution temperature for the trials.</p>
	<h4><strong>Table 1: Reaction progress</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td><strong>Trial</strong></td>
				<td><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] (M)</strong></td>
				<td><strong>[HCl] (M)</strong></td>
				<td><strong>Time (s)</strong></td>
				<td><strong>Temp (&#176;C)</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">2</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">3</td>
				<td style="text-align: center;">0.2 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">4</td>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">5</td>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">6</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">6.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">7</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">4.5 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">8</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">1.5 M</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_1._Reaction_progress.xlsx" target="_blank">Click Here to download Table 1</a></p>
	<p>We want to make sure that the reactions are all taking place at room temperature. You&#39;ll perform two benchmark trials, three trials with different sodium thiosulfate concentrations, and three trials with different HCl concentrations. You will vary the concentrations by diluting stock solutions of sodium thiosulfate and HCl, as shown in the following tables.</p>
	<h4><strong>Table 2: Sodium thiosulfate dilutions</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>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 0.2 M sodium thiosulfate</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">5 mL</td>
				<td style="text-align: center;">15 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.10 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">10 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">15 mL</td>
				<td style="text-align: center;">5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.20 M</td>
				<td style="text-align: center;">20 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_2._Sodium_thiosulfate_dilutions.xlsx" target="_blank">Click Here to download Table 2</a></p>
	<h4><strong>Table 3: HCl Dilutions</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>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 6.0 M HCl</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1.5 M</td>
				<td style="text-align: center;">2.5 mL</td>
				<td style="text-align: center;">7.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">3.0 M</td>
				<td style="text-align: center;">5.0 mL</td>
				<td style="text-align: center;">5.0 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">4.5 M</td>
				<td style="text-align: center;">7.5 mL</td>
				<td style="text-align: center;">2.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">6.0 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_3._HCl_Dilutions.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<p>Remember to use caution when handling HCl, which is toxic and highly corrosive. Sulfur dioxide, which is a gaseous product of this reaction, is also toxic. You will leave the reaction waste in the fume hood overnight to let the sulfur dioxide escape harmlessly.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-6">
		<div class="lab-step">First, put on a lab coat, splash-proof safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Fill a plastic wash bottle with deionized water and get a test tube marked with an X.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Clamp the test tube over the center of a small stir plate. The label must be on the opposite side from you. Make sure that you can clearly see the X. Place a small magnetic stir bar in the test tube.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use a thermometer clamp to secure a glass thermometer in the lower third of the tube, keeping it clear of the X and the stir bar.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Cut a sheet of about 20 squares of plastic paraffin film from a roll and bring the sheet back to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Lay paper towels in the fume hood as a clean surface for labware to be reused.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Label a 600-mL beaker as &#8216;waste&#8217; for the reaction, a 100-mL beaker as &#8216;0.2 M sodium thiosulfate&#8217;, and a 50-mL beaker as &#8216;6 M HCl&#8217;.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Fill the 100-mL beaker with 0.2 M sodium thiosulfate from the stock bottle in the dispensing hood.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">After that, bring the 50-mL beaker and a watch glass to the acid-dispensing hood to obtain 50 mL of 6 M HCl. Cover the filled beaker with the watch glass and bring your portion of HCl back to your hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Prepare 20 mL of 0.1 M sodium thiosulfate for the first benchmark trial. Use a 10-mL volumetric pipette to measure 10 mL of 0.2 M sodium thiosulfate and dispense it into a 20-mL volumetric flask. Fill the 20-mL volumetric flask with deionized water to the mark.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Then, cut a square of plastic paraffin film, seal the flask, and invert it several times to thoroughly mix the solution.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Remove the film and pour the solution into the test tube.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Prepare 10 mL of 3 M HCl. Pipette 5 mL of 6 M HCl into a 10-mL graduated cylinder. Add deionized water to fill the graduated cylinder to the 10-mL line.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Seal the top of the graduated cylinder with plastic paraffin film and thoroughly mix the HCl solution by inverting it several times.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, start the stir motor and get a reset stopwatch. Confirm that you can see the X through the solution in the test tube.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Simultaneously start the stopwatch and pour the 3 M HCl into the test tube.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Watch the X through the solution, which will start becoming cloudy when enough sulfur has been produced to saturate it. Stop the stopwatch exactly when the solution becomes so opaque that the X disappears.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Record the time in your lab notebook for benchmark trial 1 and then reset the stopwatch. Also, record the temperature of the solution.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Now, turn off the stir motor, remove the thermometer, and rinse it with deionized water into the waste beaker.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Then, empty the test tube into the waste beaker, retrieve the stir bar with long forceps, and rinse the test tube and stir bar with deionized water.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Thoroughly clean the test tube with a test tube brush. Briefly rinse the volumetric flask and graduated cylinder with deionized water as well. Dry everything with paper towels.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Repeat the trial in the exact same way for the second benchmark time. If the time is not within 3 - 5 s of the first trial, repeat the benchmark trial until you have two consistent trials.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Follow the same overall procedure for each subsequent trial, changing the dilutions as indicated in your table and cleaning your glassware between each trial. Use a 5-mL volumetric pipette as needed when measuring the sodium thiosulfate stock solution in trials four and five.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once you have performed all eight trials, rinse your glassware and tools one last time and neutralize the reaction waste with baking soda. Use caution because sulfur dioxide is one of the reaction products.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Transfer the neutralized reaction waste to the designated waste container. Then, pour any remaining sodium thiosulfate solution into its labeled waste container and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Neutralize any remaining HCl with baking soda before flushing it down the drain with tap water.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Finally, clean and put away your labware according to your standard practices and throw out any trash remaining in your hood. Your instructor will dispose of the reaction waste later.</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">Now, estimate the reaction order of sodium thiosulfate and HCl. Calculate the starting concentration of sodium thiosulfate in the reaction mixture for trials 1 through 5. Looking at the concentration of sodium thiosulfate and the reaction times, we see that as the concentration doubles, the time to opacity is halved.
		<h4><strong>Table 4: Estimating reaction order sodium thiosulfate</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>Trial</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] added</strong></td>
					<td style="text-align: center;"><strong>Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> volume (mL)</strong></td>
					<td style="text-align: center;"><strong>Total volume (mL)</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
					<td style="text-align: center;"><strong>Inverse time (s<sup>-1</sup>)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<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 style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<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 style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<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 style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<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 style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_4._Estimating_reaction_order_sodium_thiosulfate.xlsx" target="_blank">Click Here to download Table 4</a></div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Remember that each trial used the same volume, so it took the same amount of sulfur to make each solution opaque. Thus, the reaction is twice as fast when the sodium thiosulfate concentration is doubled.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">When the concentration increases by a factor of 4, the reaction rate also increases by a factor of 4. This one-to-one relationship suggests that the reaction was first order for sodium thiosulfate. If so, we would expect a plot of rate versus sodium thiosulfate concentration to be linear.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">We didn&#39;t measure how much sulfur it took to turn the solution opaque, but we assume that it was the same for each trial. This lets us use the inverse of time to opacity as a stand-in for the rate. The slope won&#39;t be equal to k, but that&#39;s OK for this lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Plot inverse reaction time as a function of sodium thiosulfate concentration. This confirms the linear relationship between sulfur production and thiosulfate concentration. Thus, the reaction seems to be first order with respect to thiosulfate.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the same method to estimate the reaction order of HCl. First, calculate the starting concentration of HCl in the benchmark trials and in trials 6 through 8.
		<h4><strong>Table 5: Estimating reaction order HCl</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>Trial</strong></td>
					<td style="text-align: center;"><strong>[HCl] added</strong></td>
					<td style="text-align: center;"><strong>HCl volume (mL) </strong></td>
					<td style="text-align: center;"><strong>Total volume (mL) </strong></td>
					<td style="text-align: center;"><strong>[HCl] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></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;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></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;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></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;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></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/50/Table_5._Estimating_reaction_order_HCl.xlsx" target="_blank">Click Here to download Table 5</a></div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Then, look at the concentration of HCl and the reaction times for these trials. The reaction times are nearly identical, so the HCl concentration has no effect on the reaction rate. This means that HCl seems to be zeroth order for this reaction.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Lastly, add up the reaction orders of the reactants to get the overall reaction order.</div>
		</li>
	</ul>
	</li>
</ol>

Concentration Dependence and Reaction Order - Procedure

Dependencia de la concentración

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

	Concentration DependenceExpand
	In this experiment, you will combine varying concentrations 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>

Técnicas Básicas de Química Orgánica

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 ...

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

Recristalización

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

	Recrystallization of AcetanilideExpand
	In this part of ...