<p class="jove_content">PAL foi financiado por um Parkinson UK Research Fellowship (grant F1002). Este trabalho foi apoiado por um novo investigador MRC Research Grant (Sr/L010933/1) e pelo programa de MRC concede senhor/N026004/1 para PAL e pela bolsa de estudo BBSRC BB/M017222/1 a jato. Agradecemos a coorte de 2014-15 de aposentado parte 1 alunos da Universidade de Reading para tomar parte na primeira iteração deste coortes prático e subsequentes para seus comentários e sugestões.</p>

<p class="jove_content">Nota: O protocolo abaixo foi desenvolvido para ser realizado ao longo de duas horas (incluindo a conclusão de planilhas de classe) em um laboratório de ensino purpose-built. As etapas experimentais foram deliberadamente colocar juntos para excluir a análise mais detalhada da cinética enzimática (realizada no âmbito deste curso usando o modelo de dados); no entanto — conforme observado na discussão — o protocolo fornece um modelo para análises mais avançadas, dependendo do tempo, instalações e o nível de realização.</p>
<p class="jove_content">SEGURANÇA: Este prático deve ser efectuado de acordo com as regras da prática boa de laboratório químico (GCLP) — equipamento de protecção pessoal, incluindo o casaco de laboratório afivelados, luvas descartáveis e óculos de segurança deve ser usado em todos os momentos no laboratório .</p>
<p class="jove_title">1. extrair a enzima</p>
<ol><li>Esmaga um comprimido de lactase, contendo 200 mg de enzima, para um mesmo pó usando um almofariz e um pilão.</li>
	<li>Coloque o pó resultante em um tubo de 15 mL rotulados de "suspensão" e ressuspender em 10 mL de soro de 100 mM tamponado fosfato (PBS).</li>
	<li>Vórtice para 1 min maximizar a extração da enzima.</li>
	<li>Transferi 1 mL da suspensão para um tubo de 1,5 mL centrífuga e centrifugar por 1 min a 10.000 x <em>g </em>de partículas sólidas de sedimentos.</li>
	<li>Transferi 500 µ l do sobrenadante para um tubo limpo 1,5 mL rotulado "Extrato da Lactase".</li>
</ol><p class="jove_title">2. observar a reação colorimétrica</p>
<ol><li>Lugar 390 µ l de PBS a 100 mM em um tubo de 1,5 mL rotulados "A reação".</li>
	<li>Adicione 100 µ l de solução ONPG 5mm e misture bem, num Vortex.<br>Atenção: Esta prática usa orto-nitrofenol -<em>beta</em>-D-galactopyranoside (ONPG) como um substituto para a lactose. Desde ONPG é um composto fenólico, devem ser manuseado com cuidado. Em caso de contacto com a pele com ONPG, a pele exposta deve ser lavada imediatamente. Todos os derramamentos devem ser limpou imediatamente com toalhas de papel para ser escoado o fluxo adequado de resíduos.</li>
	<li>Adicione 10 µ l de extrato para o tubo de reação A e misture bem, num Vortex.</li>
	<li>Observar a mistura de reação por 5 minutos e observe quaisquer alterações colorimétricas para a solução.</li>
</ol><p class="jove_title">3. medir a atividade da enzima</p>
<ol><li>Configurar dois tubos de 1,5 mL: um rotulado "Reação B", uma marcada "controle".</li>
	<li>Adicionar 390 µ l de PBS a 100 mM para a reação do tubo B, 400 µ l de PBS de 100 mM para o tubo de controle e em seguida, adicionar 100 µ l de ONPG de 5 mM a cada tubo.</li>
	<li>Misture o conteúdo num Vortex.</li>
	<li>Adicionar 10 µ l de extracto de lactase no tubo de reação B, misturar vortexing e permitir a reação proceder para 1 min à temperatura ambiente.</li>
	<li>Decorrido 1 min, adicione 500 µ l de carbonato de sódio 1 M para ambos os tubos para inibir a enzima lactase, aumentando o pH, encerrando assim a reação.</li>
	<li>Transferir 500 µ l de cada tubo em cubetas de espectrofotômetro limpo e medir a absorbância a 420 nm, utilizando o Espectrofotômetro.</li>
	<li>Observe a absorvância para reação B e amostras de controle e em seguida, subtrair o valor do controle do valor de reação para derivar a mudança de absorbância devido à hidrólise de ONPG na presença da enzima activa.</li>
</ol><p class="jove_title">4. efeito da temperatura na atividade enzimática</p>
<ol><li>Configurar o controle de três tubos e três tubos de reação conforme descrito na seção 3 e rotular estes<strong> </strong>"4 ° C", "Temperatura ambiente" e "37 ° C".</li>
	<li>Após a adição da enzima extrair, incubar um tubo no gelo, um em temperatura ambiente e um a 37 ° C (em um banho de água pré-aquecida).</li>
	<li>Permitir que as reações prosseguir por 1 min e então terminar a reação adicionando 500 µ l de carbonato de sódio 1 M para cada tubo.</li>
	<li>Medir a absorbância a 420 nm para cada tubo, conforme descrito na seção 3. Registre os valores, subtraindo o valor do controle do valor de reação em cada caso.</li>
</ol>

<ol>
	<li>Berg, J. M., Tymoczko, J. L., Gatto, J. G., Stryer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Biochemistry</em>. , (2015).</li><li>Jittam, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+seaweed+enzyme-catalyzed+bromination+of+bromophenol+red:+An+inquiry-based+kinetics+laboratory+experiment+for+undergraduates.">Red seaweed enzyme-catalyzed bromination of bromophenol red: An inquiry-based kinetics laboratory experiment for undergraduates.</a> <em style='font-style: italic'>Biochemistry and Molecular Biology Education</em>. <b>37</b>, 99-105 (2009).</li><li>Troelsen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult-type+hypolactasia+and+regulation+of+lactase+expression.">Adult-type hypolactasia and regulation of lactase expression.</a> <em style='font-style: italic'>Biochimica et biophysica acta</em>. <b>1723</b>, 19-32 (2005).</li><li>Juers, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+view+of+the+action+of+Escherichia+coli+(lacZ)+beta-galactosidase.">A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.</a> <em style='font-style: italic'>Biochemistry</em>. <b>40</b>, 14781-14794 (2001).</li><li>Plimmer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+presence+of+lactase+in+the+intestines+of+animals+and+on+the+adaptation+of+the+intestine+to+lactose.">On the presence of lactase in the intestines of animals and on the adaptation of the intestine to lactose.</a> <em style='font-style: italic'>Journal of Physiology</em>. <b>35</b>, 20-31 (1906).</li><li>Krebs, H. A., Salvin, E., Johnson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+citric+and+alpha-ketoglutaric+acids+in+the+mammalian+body.">The formation of citric and alpha-ketoglutaric acids in the mammalian body.</a> <em style='font-style: italic'>The Biochemical journal</em>. <b>32</b>, 113-117 (1938).</li><li>Vesa, T. H., Marteau, P., Korpela, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactose+intolerance.">Lactose intolerance.</a> <em style='font-style: italic'>Journal of the American College of Nutrition</em>. <b>19</b>, 165S-175S (2000).</li><li>Robayo-Torres, C. C., Nichols, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+differentiation+of+congenital+lactase+deficiency+from+adult-type+hypolactasia.">Molecular differentiation of congenital lactase deficiency from adult-type hypolactasia.</a> <em style='font-style: italic'>Nutrition Reviews</em>. <b>65</b>, 95-98 (2007).</li><li>Swallow, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+lactase+persistence+and+lactose+intolerance.">Genetics of lactase persistence and lactose intolerance.</a> <em style='font-style: italic'>Annual Review of Genetics</em>. <b>37</b>, 197-219 (2003).</li><li>Enattah, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+introduction+of+two+lactase-persistence+alleles+into+human+populations+reflects+different+history+of+adaptation+to+milk+culture.">Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture.</a> <em style='font-style: italic'>American Journal of Human Genetics</em>. <b>82</b>, 57-72 (2008).</li><li>Lowe, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rapid+detection+of+lactose+fermentation+in+paracolon+organisms+by+the+demonstration+of+beta-D-galactosidase.">The rapid detection of lactose fermentation in paracolon organisms by the demonstration of beta-D-galactosidase.</a> <em style='font-style: italic'>Journal of Medical Laboratory Technology</em>. <b>19</b>, 21-25 (1962).</li><li>Russo, S. F., Moothart, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+study+of+the+Enzyme+Lactase.">Kinetic study of the Enzyme Lactase.</a> <em style='font-style: italic'>Journal of Chemical Education</em>. <b>63</b>, 242 (1986).</li></ol>

<p class="jove_content">Os autores têm sem conflitos de interesse a divulgar.</p>

<p class="jove_content">Um detalhado conhecimento e compreensão das enzimas, reações enzimáticas e cinética enzimática é necessária para uma vasta gama de tópicos abrangendo biologia, biomedicina e farmacologia. O protocolo descrito acima usa lactase como um exemplo de uma enzima relevante para a saúde humana, para demonstrar reações enzimáticas, fornecendo as competências-chave que podem ser usadas em experimentos de laboratório-baseado mais avançados.</p>
<p class="jove_content">Este protocolo foi deliberadamente concebido para ser tão robusto como possíveis, permitindo que os estudantes com pouca ou nenhuma experiência de laboratório molhado para produzir resultados interpretáveis em um curto espaço de tempo. No ano letivo de 2014/2015 da faculdade de farmácia da Universidade de Reading, um total de 144 estudantes, organizados em grupos de 3, realizado o experimento "medindo lactase", com todos os grupos capazes de detectar algum nível de atividade enzimática de tablet de lactase extratos.</p>
<p class="jove_content">Há uma série de passos críticos no presente protocolo. Em primeiro lugar, é essencial que a extração inicial seja eficiente, permitindo que a solubilização de suficiente enzima activa para análises subsequentes. Em nossa experiência, primeiro ano estudantes podem alcançar isto seguindo o protocolo descritos; no entanto algumas orientações de manifestantes de laboratório era necessária neste momento. Embora parece um ponto óbvio, um fator chave no sucesso deste protocolo é a capacidade de cuidadosamente medir volumes, corretamente rotular tubos e siga as instruções. Enquanto um certo nível de proficiência pode ser assumido por muitos alunos, monitoramento cuidadoso e um pedido claro para que os alunos pedir ajuda no caso em que eles não tem certo de o que fazer em seguida fornece a maior probabilidade de um ensaio bem sucedida.</p>
<p class="jove_content">Avaliação de estudante pode ser efectuada em-classe planilha (disponível como material suplementar), pedindo os alunos observar dados de experimentos, comentário sobre seus resultados e link para informações de plano de fundo/leitura do material da palestra, ou por mais detalhada, sem avaliação de classe com o modelo de dados fornecido permitindo análises cinéticas ser tentada e avaliada.</p>
<p class="jove_content">O protocolo apresentado aqui é um exemplo simples de um ensaio de atividade de lactase sob condições de laboratório de ensino, e há muitas oportunidades para aumentar a complexidade do experimento para permitir análises mais avançadas. Em particular, o protocolo apresentado fornece uma introdução ao conceito de cinética enzimática, mas não permite a análise cinética de dados experimentais. Como tal, recomendamos Visualizar o protocolo descrito como um modelo inicial para classes, com os detalhes precisos, adaptados para atender os objetivos específicos e talentos da coorte estudante realizar os experimentos. Exemplos de experimentos prolongados poderiam incluir: repetidas medições em diferentes temperaturas, usando concentrações de substrato diferentes para permitir a análise estatística, análise cinética de dados (apropriados para estudantes de bioquímica/majors), começando com vários comprimidos diferentes e permitindo que os alunos a avaliar diferente atividade da enzima em cada um (com a adição de tabuletas controle, apropriadas para estudantes de ciência forense/majors), avaliação do impacto de desnaturação térmica ou realização de competição experiências por adição de lactose ou inibidores de lactase (<strong>tabela 1</strong>). Um protocolo detalhado para análise cinética de lactase já havia sido publicado<sup class="xref">12</sup>.</p>
<p class="jove_content">Uma força importante deste protocolo é que ele combina uma introdução básica à cinética de enzimologia e enzima com um estudo de caso da vida real da enzimologia na medicina. Isso faz com que este laboratório experiência de particular relevância para os estudantes de medicina, farmácia, ciências biomédicas e assuntos relacionados. Importante, o fato de que haja uma condição médica séria e uma questão dietética mais generalizada associada com catabolismo à lactose fornece a oportunidade de criar uma vasta gama de questões éticas e médicas com os alunos. Isto poderia incluir as questões que envolvem testes genéticos para condições médicas e associado a discussões relacionadas a terapia genética e aconselhamento genético, bem como a utilização e venda de suplementos medicinais. Uma limitação importante é que o protocolo não permite a estimativa precisa da concentração de enzima, devido às matérias-primas usadas para a extração. Uma modificação para dar conta disto, no entanto, seria usar a enzima de grau reagente para o ensaio. Importante, isto também permitiria um cálculo mais preciso da cinética para a enzima lactase.</p>
<p class="jove_content">Em conclusão, análise da atividade da lactase em um laboratório de ensino ambiente fornece uma introdução robusta, atraente e interessante para o campo da biologia de enzima para fase inicial estudantes universitários.</p>

<p class="jove_content">As enzimas são um tipo especializado de proteína que agem como catalisadores biológicos, por reações químicas na vida organismos<sup class="xref">1</sup>. A ação de enzimas é fundamental para a vida, fornecendo energia, eliminação de resíduos e permitindo que os organismos funcionar. Enzimas de compreensão é, portanto, fundamental para uma compreensão completa da vida. Esse conhecimento é essencial para uma grande variedade de programas de grau de nível de Universidade, variando de ciências médicas de biologia. Enquanto um fundo detalhado desde princípios de catálise até modelos teóricos da atividade da enzima pode ser fornecido aos estudantes utilizando palestras e material de leitura, as propriedades de reacções enzimáticas são melhor compreendidas pelas mãos na experiência prática de enzimas em ação como previamente demonstraram<sup class="xref">2</sup>. Este protocolo fornece um simples para seguir o paradigma experimental para medir a atividade da enzima em condições de laboratório, utilizando lactase como um exemplo de uma enzima com uma actividade relevante para a saúde e nutrição humana.</p>
<p class="jove_content">A lactase hidrolase glicosídica (CE-3.2.1.23/26) é uma enzima de central importância nutricional para mamíferos<sup class="xref">3</sup>. A atividade da lactase é altamente conservada através da evolução e deriva da família de enzimas beta-galactosidase — uma família presente de <em>Escherichia coli</em> através de<em> Homo sapiens</em> (<strong>Figura 1</strong>, PDB 1JZ8)<sup class="xref">4</sup>. A importância fundamental da lactase num cenário nutricional humano decorre de seu papel em permitir que a quebra da lactose em seus componentes monossacarídeos constituintes, que podem ser usados para gerar energia no corpo. Lactase catalisa a hidrólise da ligação glicosídica em lactose dissacarídeo, liberando a galactose e a glicose (<strong>Figura 2</strong>)<sup class="xref">5</sup>. Esses monossacarídeos são usados principalmente para a geração de adenosina trifosfato (ATP) <em>através </em>do ciclo do ácido cítrico e fosforilação oxidativa<sup class="xref">6</sup>. Durante o desenvolvimento infantil e neonato, lactase é altamente expresso no sistema digestivo humano, quebrando à lactose recebida do leite materno de que lactose é o componente primário de hidrato de carbono e uma das principais fontes de nutrição durante os primeiros anos<sup class="xref"> 7</sup>. a importância médica de lactase é destacada por deficiência de lactase congênita (CID), uma rara condição autossômica recessiva, causada por mutações no gene da lactase (<em>LCT</em>) que codifica para a enzima de lactase<sup class="xref">8</sup>. Recém-nascidos com CID apresentam pouca atividade de lactase, assim eles não podem ser alimentados com leite materno, qualquer outro tipo de leite ou fórmula que contenha lactose.</p>
<p class="jove_content">Durante a infância, expressão de lactase normalmente é reduzida; no entanto, esta redução após o desmame varia geograficamente, com aproximadamente 35% dos adultos em todo o mundo continua a expressar a enzima<sup class="xref">9</sup>. Expressão sustentado de lactase, conhecido como persistência da lactase, permite que indivíduos continuar a digerir leite e produtos lácteos de uma variedade de fontes. Por outro lado, a perda de expressão de lactase pode levar à intolerância à lactose, também conhecido como adulto-tipo hypolactasia (ATH), resultante de uma incapacidade de quebrar a lactose no intestino. ATH é caracterizada por uma compilação acima de lactose no cólon após a ingestão de lactose, contendo produtos alimentares. No cólon, a lactose acumulada é fermentada pela fauna microbiana do intestino, liberando gases incluindo hidrogênio, metano e dióxido de carbono. A produção desses gases em indivíduos com deficiências de enzima lactase promover distensão abdominal, flatulência aumentada, dor, náusea e borborygmi (rumbling estômago)<sup class="xref">7</sup>. Aumento dos níveis de lactose no tracto digestivo também pode levar a fezes soltas.</p>
<p class="jove_content">O controle da expressão do gene <em>LCT</em> é modulado por polimorfismos localizados em intrões do gene <em>MCM6</em> nas proximidades. Indivíduos com expressão sustentado de lactase carregam polimorfismos que funcionam como potenciadores distais fortes para a expressão do gene <em>LCT</em> , compensando assim o normal no Regulamento da transcrição de <em>LCT</em> durante o desmame e, consequentemente, sustentar a expressão de lactase na idade adulta<sup class="xref">3</sup>. Polimorfismos de potenciador sugeridos que foram selecionadas positivamente após a domesticação do gado e camelos no Oriente Médio há mais de cinco mil anos de<sup class="xref">9</sup><sup>,</sup><sup class="xref">10</sup>.</p>
<p class="jove_content">Os sintomas resultantes de ATH podem ser gerenciados por reduzir a ingestão de lactose, por exemplo, removendo produtos lácteos da dieta. Uma abordagem alternativa para ATH e a abordagem de escolha para CID, é o uso de suplementos de lactase, amplamente disponíveis em farmácias. Estes suplementos fornecem lactase isolado de uma variedade de fontes, incluindo leveduras e bactérias, em um formulário líquido ou comprimido-based que pode ser tomado com ou adicionado à lactose contendo alimentos. O suplemento vai hidrolisar uma proporção de lactose presente nos alimentos de produtos de glicose e galactose, assim permitindo a sua absorção e evitar o acúmulo de substrato de lactose não digerida no intestino.</p>
<p class="jove_content">Baseado no uso de suplementos de lactase, como uma dieta ajuda, nós desenvolvemos uma experiência de laboratório de enzimologia simples apropriada para primeiro ano biomédica ciência ou farmácia estudantes. Este experimento de laboratório aproveita-se de suplementos de lactase comercialmente disponível e usa orto-nitrofenol -<em>beta</em>-D-galactopyranoside (ONPG) para fornecer um ponto de extremidade colorimétrico para medir a clivagem das ligações glicosídicas por lactase (<strong> Figura 3</strong>)<sup class="xref">11</sup>. ONPG atua um substrato artificial para a lactase, que quando submetidos à hidrólise por esta enzima produz a D-galactose e orto-nitrofenol. O último produto tem uma cor amarela, absorve luz no comprimento de onda de 420 nm. Através da quantificação quaisquer alterações em absorvância a 420 seguinte nm a exposição de ONPG a lactase, é possível estimar a atividade desta enzima. Este experimento de laboratório fornece uma demonstração de atividade enzimática hidrolase. Construindo em repetições adicionais e realização de ensaios em diferentes condições, é possível incorporar mais sofisticadas análises de cinética enzimática, proporcionando um valioso exemplo de vida real de enzimas em ação relevante para a saúde humana.</p>

<table><tbody><tr><td>Lactase tablet</td><td>Lamberts</td><td>8511-60</td><td></td></tr><tr><td>Phosphate buffered saline (powdered)</td><td>Sigma</td><td>P3813</td><td>Dissolved in deionized water to a final concentration of 100 mM</td></tr><tr><td>ONPG</td><td>Sigma</td><td>N1127</td><td>Dissolved in deionized water to a final concentration of 5 mM</td></tr><tr><td>Sodium Carbonate</td><td>Sigma</td><td>451614</td><td>Dissolved in deionized water to a final concentration of 1 M</td></tr><tr><td>De-ionized water</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Pestle and mortar</td><td>VWR</td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Jenway 6315</td><td></td><td></td></tr><tr><td>Pipettes</td><td>Gilson</td><td></td><td></td></tr><tr><td>15 mL tubes</td><td>VWR</td><td></td><td></td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Spectrophotometer cuvettes</td><td>Jenway</td><td></td><td></td></tr><tr><td>Vortex</td><td>Vortex genie 2</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td></td><td></td></tr><tr><td>Ice bucket</td><td>VWR</td><td></td><td></td></tr><tr><td>Water bath</td><td>Thermo-Scientific</td><td></td><td></td></tr><tr><td>Weighing scales</td><td>Thermo-Scientific</td><td></td><td></td></tr></tbody></table>

measuring lactase enzymatic activity in the teaching lab

<p class="jove_content">Compreender como funcionam as enzimas e isto aos exemplos da vida real, são fundamental para uma ampla gama de graduação em ciências biológicas e biomédicas. Tão fácil de seguir protocolo foi desenvolvido para estudantes de graduação de farmácia primeiros ano e fornece uma introdução de nível básico de reacções enzimáticas e procedimentos analíticos para análise enzimática. A enzima de escolha é a lactase, como este representa um exemplo de uma enzima disponível comercialmente relevante para a prática de doença humana/farmacêutica. Lactase é extraído de comprimidos de suplemento dietético e avaliados usando um ensaio colorimétrico baseado a hidrólise de um substrato artificial para lactase (Orto-nitrofenol -<em>beta</em>-D-galactopyranoside, ONPG). Lançamento do Orto-nitrofenol após a clivagem hidrolítica de ONPG pela lactase é medido por uma alteração na absorbância a 420 nm e o efeito da temperatura sobre a reação enzimática é avaliada por realizar a reação no gelo, à temperatura ambiente e em 37 ° C. Análise mais avançada pode ser implementada usando este protocolo, avaliar a atividade da enzima em condições diferentes e usando diferentes reagentes.</p>

<p class="jove_content" fo:keep-together.within-page="1">Resultados representativos para a seção 2 do protocolo são mostrados na <strong class="xfig">Figura 4A</strong>. A reação de controle, na ausência da enzima lactase, permanece clara enquanto a solução de reação, contendo extrato do comprimido de lactase, fica amarela como ONPG é hidrolisado liberando <em>orto</em>- nitrofenol. <strong class="xfig">Figura 4B</strong> mostra a quantificação utilizando unidades arbitrárias da secção 4 do protocolo, seguir análise de amostras usando o Espectrofotômetro. Produção de orto-nitrofenol é mais baixa quando a reação é incubada no gelo e mais alto quando a reação é realizada a 37 ° C.</p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54377/54377fig1.jpg"><br><strong class="xfig">Figura 1</strong> <strong>:</strong> <em><strong>Escherichia coli </strong></em> <strong>beta-galactosidase.</strong> (<strong>A</strong>) estrutura de cristal da beta-galactosidase de <em>Escherichia coli</em>, mostrando a estrutura tetramérica da ativo complexo. (<strong>B</strong>) Allolactose (mostrado em vermelho) ligado ao sítio ativo da beta-galactosidase. Imagens geradas a partir de PDB 1JZ8<sup class="xref">4</sup>. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54377/54377fig2.jpg"><br><strong class="xfig">Figura 2</strong> <strong>:</strong> <strong>Acção enzimática de lactase.</strong> Hidrólise da lactose por lactase para produzir beta-D-galactose e beta-D-glicose. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54377/54377fig3.jpg"><br><strong class="xfig">Figura 3</strong> <strong>:</strong> <strong>Hidrólise de orto-nitrofenol -</strong> <em><strong>beta</strong></em> <strong>-D-galactopyranoside.</strong> Hidrólise ONPG pela lactase para produzir beta-D-galactose e orto-nitrofenol (que é na cor amarela), que permite a estimativa da atividade da lactase de medição de absorbância a 420 nm. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig3large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> </p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/54377/54377fig4.jpg"><br><strong class="xfig">Figura 4</strong> <strong>:</strong> <strong>Hidrólise ONPG.</strong> Resultados representativos de hidrólise ONPG, mostrando tubos de controle e reação (<strong>A</strong>) e dados representativos da secção 4 do protocolo registrados em planilhas em sua classe, mostrando a absorvância cru em unidades arbitrárias em 420 nm, corrigida para o fundo e em três temperaturas diferentes (<strong>B</strong>). <a href="https://www.jove.com/files/ftp_upload/54377/54377fig4large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a></p>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td><strong>Condição experimental</strong></td>
			<td><strong>Variáveis experimentais</strong></td>
		</tr>
<tr>
<td>Temperatura</td>
			<td>0 ° C, 10 ° C, 20 ° C, 30 ° C, 50 ° C</td>
		</tr>
<tr>
<td>Concentração de substrato</td>
			<td>0 mM, 1 mM, 5 mM, 10 mM ONPG</td>
		</tr>
<tr>
<td>Inibição competitiva</td>
			<td>à lactose ONPG além de 0 mM, 1 mM, 5 mM ou 10 mM 5 mM</td>
		</tr>
<tr>
<td>Dependência do tempo</td>
			<td>0 s, 15 s, 30 s, 1 min, 5 min, 10 min, 20 min</td>
		</tr>
<tr>
<td>Desnaturação térmica</td>
			<td>Extrato da lactase aquecido a 100 ° C durante 10 min antes de ensaio</td>
		</tr>
</tbody></table><p class="jove_content" fo:keep-together.within-page="1"><strong>Tabela 1: potencial estendido condição experimental</strong>. Sugere-se experiências estendidas, a efectuar em triplicado, investigar aspectos específicos da biologia do lactase.</p>

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Measuring Lactase Enzymatic Activity in the Teaching Lab

<p class="jove_content">A atividade enzimática da lactase é essencial para o processamento catabólico de lactose o dissacarídeo. Aqui, a atividade da lactase encontrado em suplementos dietéticos é analisada usando um ensaio colorimétrico. Isto fornece aos alunos uma plataforma experimental para a compreensão da atividade da lactase e enzima cinética.</p>

Medindo a atividade enzimática de Lactase no laboratório de ensino

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112.9K visualizações.  University of Reading. A atividade enzimática da lactase é essencial para o processamento catabólico de lactose o dissacarídeo. Aqui, a atividade da lactase encontrado em suplementos dietéticos é analisada usando um ensaio colorimétrico. Isto fornece aos alunos uma plataforma experimental para a compreensão da atividade da lactase e enzima cinética.

Vídeo: Measuring Lactase Enzymatic Activity in the Teaching Lab

Lab Techniques

<h4>Measurement Accuracy</h4>
<p>The ability to repeat an experiment and get the same results, or <em>reproducibility</em>, is essential in scientific research. However, it&#8217;s impossible to repeat an experiment if you don&#8217;t know how you did it. Thus, scientists keep detailed records of their experiments in lab notebooks. These records include important information like the amount of each material used and descriptions of each step of the procedure.</p>
<p>Equipment, like balances and volumetric or graduated glassware, is used to measure solid and liquid compounds for experiments. The precision of each measurement is limited by the equipment used. Balances and other equipment with digital readouts automatically display values at the maximum precision that they can support. The precision of graduated glassware and rulers depends on the increment, which is the amount represented by the smallest distance between two marks.</p>
<p>The surface of most liquids curves upwards at the edges in glass containers. This type of curved liquid surface is called a concave meniscus. When the edges of the liquid curve down, it is called a convex meniscus. When measuring liquid volumes, the reference point for the top of the liquid is the bottom of a concave meniscus or the top of a convex meniscus. To accurately identify the position of the meniscus, liquid volumes are read by looking at the liquid&#8217;s surface from the side at eye-level. Looking at the liquid from above or below will cause the liquid surface level to appear higher or lower than it actually is.</p>
<p>When the reference point of the meniscus touches a mark on graduated or volumetric glassware, the glassware is holding the volume defined for that mark. Volumetric glassware is designed to measure a specific volume, so it only has one mark. Graduated glassware is designed to measure a range of volumes, so it has many marks, or <em>graduations</em>, which are labeled at regular intervals. Both graduated and volumetric glassware may have additional manufacturer-provided information about their precision.</p>
<h4>Calibration and Uncertainty</h4>
<p>Manufacturers calibrate equipment to ensure that the measurements are accurate to within a specified range of uncertainty. For example, a high-precision analytical balance may read out to four decimal places (0.0000 g) with an uncertainty of &#177; 0.0001 g. A reading of 0.0345 g indicates that the true value is between 0.0344 g and 0.0346 g.</p>
<p>Glassware typically has its measurement uncertainty printed on it. To accurately record volume measurements, you must consider both the increment and the uncertainty. For example, a precise volume measurement in a 100 mL graduated cylinder with 1 mL increments and an uncertainty of &#177; 0.5 mL would be recorded to the tenths place (000.0 mL &#177; 0.5 mL). If the reference point of the meniscus falls between two marks, you should estimate a value for the tenths place; otherwise, leave it as 0. Since temperature affects volume, the temperature for which the glassware is calibrated will be printed on it.</p>
<p>Glassware is calibrated either <em>to contain</em> (TC) or <em>to deliver</em> (TD). TC glassware holds the specified volume of liquid when it is filled to the mark, but a small amount of liquid will be left behind when it is poured into another container. Solutions are often prepared in TC glassware because the accuracy of the solution volume affects the accuracy of the concentration. Volumetric flasks are typically calibrated to contain.</p>
<p>TD glassware holds a little more than the amount of liquid specified when it is filled to the mark, but it dispenses only the specified volume. Thus, TD glassware should not be emptied completely when dispensing liquids. This type of glassware is useful for transferring a precise volume of liquid to another container. Both graduated and volumetric pipettes are typically calibrated to deliver.</p>
<p>Unless otherwise specified, the uncertainty of a number is assumed to be on the scale of the last digit. Thus, it&#8217;s particularly important to report the uncertainty of a measurement if the uncertainty is on a larger scale. For rulers and graduated glassware without a known uncertainty, the uncertainty is estimated as half of the smallest increment. The uncertain digit is based on where the meniscus falls between two tick marks.</p>
<h4>Significant Figures</h4>
<p>Significant figures are the numbers in a value that are <em>meaningful</em>, or essential for expressing that value with the appropriate precision. All numbers other than leading zeroes (<strong>0.00</strong>1), trailing zeroes (1,<strong>000</strong>), or scientific notation multipliers (<strong>10</strong><sup>x</sup>) are always significant.</p>
<p>Leading zeroes are never significant because they can be removed by rewriting the number either in scientific notation or, if it is an SI unit, in smaller units. For example, there are only three significant figures in the value 0.00123 m because it can be rewritten as either 1.23 &#215; 10<sup>-3</sup> m or 1.23 mm without losing any information. Note that the scaling factor between different multiples of the same SI unit does <em>not</em> affect the number of significant figures.</p>
<p>Trailing zeroes are significant when they are before or after a decimal point. For example, the measurement 100.110 mL has six significant figures and 100.0 mL has four. Trailing zeroes in a number written without a decimal point are assumed to be insignificant unless they are marked otherwise. There are several different conventions for marking significant zeroes in those cases, but to avoid ambiguity it is advisable to rewrite those values in scientific notation. If the measurement is in SI units, use larger units.</p>
<p>The precision of a measurement limits the precision of any values calculated from it. A calculated value has the same number of significant figures as the <em>least</em> precise measurement or value used in its calculation. If the result has too many significant figures, it is rounded to the appropriate precision. If the result has too few significant figures, it is extended with significant trailing zeroes to preserve the precision. The significant figures of experimentally derived values, like molar mass, density, or some unit conversion factors, must also be considered when they are used in calculations.</p>
<p>The number of significant figures in a calculated value is <em>not</em> limited by physical or mathematical constants in formulas or exact numbers, like the number of data points acquired in an experiment. For example, the formula for calculating the volume of a sphere is V = 4/3&#960;r<sup>3</sup>. The number of significant figures in the calculated volume is <em>only</em> affected by the number of significant figures in the radius measurement <strong>r</strong>. 4/3&#8201; and &#960; are constants, and <sup>3</sup> is simply notation for a mathematical operation.</p>
<p>Defined conversion factors are also treated as constants. For instance, the inch is defined as exactly 25.4 millimeters; therefore, the number of significant figures of the converted value depends only on the original measurement. Always check whether the conversion factor is exactly defined or an experimental value when converting between units.</p>
<p>For simple addition and subtraction of measurements, we use significance arithmetic to determine the number of significant figures. In this case, the answer has as many decimal places as the measurement with the fewest decimal places <em>regardless of the number of significant figures</em>. Thus, 15,643.7 mL + 0.613 mL = 15,644.3 mL.</p>
<p>For multi-step calculations or complex equations that you break into smaller parts to solve, you should keep at least one or two insignificant figures on the intermediate values. For example, if you calculate the values of the numerator and denominator of a fraction separately before dividing them, you should retain the insignificant figures of the numerator and denominator when you divide them. This minimizes <em>rounding error</em>, which is the difference from the number you would get if you entered the entire formula into your calculator at once. If you write down these intermediate values, you should note which digits are insignificant.</p>
<h4>References</h4>
<p>Harris, D.C. (2015). <em>Quantitative Chemical Analysis</em>. New York, NY: W.H. Freeman and Company.</p>

Accuracy, Precision, Uncertainty, and Significant Figures - Concept

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Measurement Accuracy
The ability to repeat an experiment and get the same results, or reproducibility, is essential in scientific research. However, ...

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Scientific Measurement and Lab Skills

<p><strong>Two-Dimensional Graphs</strong></p>
<p>Some chemistry experiments take the form of directly changing one property of the system that you are studying otherwise known as the independent variable, like temperature, and measuring the effects on another property, otherwise known as the dependent variable, like volume. Once the data have been collected, the interaction between the two parameters must be <i>quantified</i> &#8212; or converted into a form that can be evaluated &#8212; and compared to other relationships.</p>
<p>Two-dimensional graphs can be used to derive certain types of mathematical relationships between two properties or to establish that no such relationship exists between them. The analysis will ultimately determine how the dependent variable changes in response to the independent variable. In the example of adjusting the temperature of a liquid or gas and monitoring changes in its volume, the temperature is the independent variable, and the volume is the dependent variable.</p>
<p>To make a two-dimensional graph, each data point must have a value known as the coordinate for both the dependent and independent variables. The independent variable is plotted on the x-axis, and the dependent variable is plotted on the y-axis. These plots are easily made in spreadsheet software, which can also be used to analyze the plotted data.</p>
<p><strong>Curve Fitting</strong></p>
<p>Once a data set has been plotted on a two-dimensional graph, curve fitting can be used to generate an equation, or <i>function</i>, for the dependent variable in terms of the independent variable. Functions represent a mathematical model that best models the data from which it is derived. Curve fitting is the technique of finding a function that produces a line that is a good match for the pattern of data points. Spreadsheet software has various curve-fitting tools, which is referred to as &#8216;best-fit&#8217;. This is usually a linear least-squares regression analysis, though most software also offers nonlinear least-squares regression.</p>
<p>The precision of the best-fit linear equation can be checked by plugging in the x values for the data points and comparing the &#8216;theoretical&#8217; results of the equation to the actual y values of the data points. Spreadsheet software will typically calculate the coefficient of determination (R<sup>2</sup>) value for the function, which shows how well the function matches the data points. The closer the R<sup>2</sup> value is to 1, the better the fit for a linear regression. Other functions have more specialized methods to determine how well the fit of the function is to the data.</p>
<p>Determining the uncertainty of dependent values calculated from the best-fit function would require complicated &#8220;error propagation&#8221; techniques. However, it is possible to calculate uncertainty within the equation in the form of the standard deviation for both the slope and y-intercept of a best-fit function. This is usually performed with a different tool than the one used to generate a two-dimensional plot.</p>
<p><strong>Standard Deviation</strong></p>
<p>The standard deviation describes the amount of variation present in a set of values. The population standard deviation (&#963;) is used when there is data from each member of a finite population, such as the mass of each marble in a bag of marbles. The sample standard deviation (<i>s</i>) is used for all other cases and is the default standard deviation calculation in spreadsheet software.<sup>1</sup> You can assume that &#8216;standard deviation&#8217; refers to the sample standard deviation.</p>
<p>Random measurement error is assumed to follow a roughly &#8216;normal&#8217; distribution, where about 68% of a set of values lie within a range of one standard deviation on either side of the mean, 95% of the values lie within two standard deviations on either side of the mean, and 99.7% of the values lie within three standard deviations on either side of the mean. Thus, the standard deviation is a useful way to describe error and uncertainty.</p>
<p>The equation for the sample standard deviation is:</p>
<p><img src="https://www.jove.com/CDNSource/lm/labs/42/42_Concepts_1.jpg" /></p>
<p>In this equation, N is the number of values; <img src="https://www.jove.com/CDNSource/lm/labs/42/30034eq02.jpg" /> is the average (or mean) of the values; and x<sub>i</sub> represents each individual value. Thus, to calculate the standard deviation by hand, calculate the mean of the set of values, subtract the mean from each value, square each difference, add the squared differences, divide the total sum by one less than the number of values, and take the square root of the quotient. The closer <i>s</i> is to zero, the less variation there is between the values. If the values are entered into spreadsheet software, the standard deviation can be calculated from within the software.</p>
<p>The number of significant figures in a standard deviation depends on what values it is for. When reporting the standard deviation for a group of data points taken under the same conditions, the appropriate number of significant figures in the mean value must first be determined. The standard deviation is then rounded to the same number of decimal places as the mean. For a set of volumes with four significant figures, a mean of 15.361 mL, and a standard deviation of 0.2313, the mean and standard deviation would be reported as 15.36 mL &#177; 0.23 mL.</p>
<p>When reporting the standard deviation of the mean and y-intercept for a best-fit function determined by least-squares analysis, which is the usual method for spreadsheet software, the first decimal place of the standard deviation is the last significant figure of the mean or y-intercept. Thus, the standard deviation should be rounded to one significant decimal place, and the slope or y-intercept should be rounded to the corresponding decimal place. For example, if the slope is 0.1691 L&#183;K<sup>-1</sup> and it has a standard deviation of 0.00512, the slope should be reported as 0.169 L&#183;K<sup>-1</sup> &#177; 0.005 L&#183;K<sup>-1</sup>.</p>
<p>If the standard deviation of the slope or y-intercept is so much smaller than its corresponding value that following this rule would give more significant figures to the slope or y-intercept than the original measurement data would allow, then instead determine the significant figures of the slope or y-intercept from the x and y values and round the standard deviation to one significant figure. Thus, for a slope of 0.1691 L&#183;K<sup>-1</sup>, a standard deviation of 0.0000512, and x and y values with four significant figures, the slope should be reported as 0.1691 L&#183;K<sup>-1</sup> &#177; 0.00005 L&#183;K<sup>-&#8205;1</sup>. In this case, it&#8217;s fine for the calculated value and its standard deviation to have a different number of decimal places. Keep in mind that the least-squares method uses both the x and y values to calculate the slope and y-intercept.</p>
<p>When reporting standard deviation as an uncertainty range, be sure to note specifically that the uncertainty represents one standard deviation. This tells the reader that there is about a 68% chance that the true value of a measurement falls within that range of the mean, assuming a normal distribution. It may often be more suitable to report uncertainty as two standard deviations from the mean, as that increases the probability to about 95%. To do this, simply multiply the standard deviation by two before rounding it to the appropriate number of significant figures.</p>
<p><strong>References</strong></p>
<ol style="font-size:17px">
	<li style="font-size:17px">Harris, D.C. (2015). <i>Quantitative Chemical Analysis</i>. New York, NY: W.H. Freeman and Company.</li>
</ol>

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

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Two-Dimensional Graphs
Some chemistry experiments take the form of directly changing one property of the system that you are studying otherwise known as ...

Acid and Base Concentrations

<h4>Acids and Bases</h4>
<p>An Arrhenius acid produces hydrogen ions when it is dissolved in water:</p>
<p style="text-align: center;">HA + H<sub>2</sub>O &#8594; H<sup>+</sup><sub>(aq)</sub> + A<sup>-</sup><sub>(aq)</sub></p>
<p>Here, HA is the non-dissociated acid, H<sup>+</sup> is the hydrogen cation, and A<sup>-</sup> is the solvated anion &#8212; called the conjugate base. An Arrhenius base produces hydroxide ions when dissolved in water:</p>
<p style="text-align: center;">BOH + H<sub>2</sub>O &#8594; B<sup>+</sup><sub>(aq)</sub> + OH<sup>-</sup><sub>(aq)</sub></p>
<p>Here, BOH is the non-dissociated base, OH<sup>-</sup> is the hydroxide ion, and B<sup>+</sup> is the solvated cation &#8212; called the conjugate acid. A conjugate base is formed when an acid loses a hydrogen ion and has the potential to gain a hydrogen. The same follows for a conjugate acid, which is formed when a base loses a hydroxyl group and has the potential to regain it. Every acid has a conjugate base, and every base has a conjugate acid.</p>
<h4>pH</h4>
<p>pH is the degree of acidity of the solution and is a measure of the amount of hydrogen ions in a solution. The pH scale is logarithmic and runs from 0 to 14; aqueous solutions with a pH below 7 are described as acidic, and aqueous solutions with a pH above 7 are described as alkaline or basic. Solutions at pH 7 are considered neutral.</p>
<p>The pH of a solution is equal to the negative log base ten of the concentration of hydrogen ions in solution.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_3.jpg" /></p>
<p>Water interacts strongly with the hydrogen ion because its strong positive charge attracts the negative pole of surrounding water molecules. In fact, they interact so strongly that they form a covalent bond and the H<sub>3</sub>O<sup>+</sup> cation, called hydronium. The above equation is rewritten to reflect this.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_4.jpg" /></p>
<p>For simplicity, we&#8217;ll refer to the concentration of hydrogen ions instead of hydronium ions when discussing pH. The lower the pH value of a solution, the more hydrogen ions that are present, and by extension, the more acidic the solution. For example, the pH of 1 mM of sulfuric acid is 2.75, whereas the pH of 1 mM of hydrochloric is 3.01. The concentration of hydrogen ions in the sulfuric acid solution is calculated as 1 &#215; 10<sup>-2.75</sup>, whereas the concentration of hydrogen ions in the hydrochloric acid solution is 1 &#215; 10<sup>-3.01</sup>. Thus, there are more hydrogen ions present in sulfuric acid, and it is more acidic. Remember, even though the pH of two solutions may vary by as little as half a pH value, due to the logarithmic nature of the pH scale, the amount of hydrogen varies greatly.</p>
<h4>Strength of Acids and Bases</h4>
<p>An acid&#8217;s strength is affected by the electronegativity of the conjugate base and the polarity of the acidic hydrogen. Strength, therefore, refers to how readily the hydrogen cation (H<sup>+</sup>) disassociates from the anion. Strong acids and bases dissociate entirely in aqueous solutions, whereas weak acids and bases only dissociate partially into their conjugate ions.</p>
<p>The dissociation constant, K<sub>a</sub>, represents acid strength. K<sub>a </sub>is calculated using the concentrations of the non-dissociated acid HA, and the concentrations of the hydrogen cations and the conjugate base, A<sup>-</sup>. Higher K<sub>a</sub> values represent stronger acids, whereas smaller K<sub>a</sub> values represent weaker acids.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_5.jpg" /></p>
<p>K<sub>a</sub> is numerically very small, and it is often reported in the form of pK<sub>a</sub>, which is the negative log base ten of K<sub>a</sub>. Lower pK<sub>a</sub> values correspond to a stronger acid, whereas higher pK<sub>a</sub> values correspond to a weaker acid.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_6.jpg" /></p>
<p>Some acids dissociate only one hydrogen ion and therefore have one pK<sub>a</sub> value. These acids are called monoprotic. However, some acids can dissociate more than one hydrogen ion and are called polyprotic. These acids have a pK<sub>a</sub> value for each hydrogen ion dissociation.</p>
<p>pK<sub>a</sub> can also be used to calculate the equilibrium pH of an acid-base reaction, as shown in the Henderson-Hasselbalch equation.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_7.jpg" /></p>
<p>The Henderson-Hasselbalch equation is used to calculate pH, when the concentrations of the conjugate base and the weak acid are known, or to calculate the pK<sub>a</sub> if the pH and concentrations are known.</p>
<h4>Titration</h4>
<p>Acid-base reactions are quantitatively studied using titration. In a titration experiment, a solution of a known concentration, called a standard solution, is used to determine the concentration of another solution. For acid-base titrations, a standardized solution of base is slowly added to an acid of unknown concentration (or the acid is added to the base). The acid-base reaction is a neutralization reaction, which forms a salt and water. When the moles of hydrogen ions in the acid are equal to the moles of hydroxyl ions added from the base, the solution reaches neutral pH.</p>
<p>To perform an acid-base titration, the standardized base is slowly added to a stirring flask of the unknown acid using a burette, which enables the measurement of volume and the dropwise addition of base. The pH of the solution is closely monitored throughout the titration using a pH indicator added to the acid. Typically, phenolphthalein is used as the solution remains colorless until it becomes basic, turning a light pink.</p>
<p>As the titration approaches the equivalence point, which is when the moles of hydrogen ions equal the moles of hydroxyl ions added, the pH indicator temporarily changes color due to an excess of hydroxyl ions. When the flask is swirled, the pH indicator&#8217;s acidic color returns. The titration is complete and has reached its endpoint when a tiny excess of hydroxyl ions changes the indicator permanently to its basic color.</p>
<p>The titration curve is a plot of the pH of a solution versus the volume of standardized base added. The equivalence point is located at the inflection point of the curve, and it is calculated as the second derivative of the titration curve.</p>
<p>If an acid is polyprotic, it will have multiple equivalence points, one for each hydrogen ion dissociation. The pH at the halfway point to the equivalence point for monoprotic acids, or between equivalence points in the case of polyprotic acids, is equal to the pK<sub>a</sub> of the acid.</p>
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). <em>Chemistry and Chemical Reactivity.</em> Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silberberg, M.S. (2009). <em>Chemistry: The Molecular Nature of Matter and Change.</em> Boston, MA: McGraw-Hill.</li>
	<li style="font-size:17px">Harris, D.C. (2015). <em>Quantitative Chemical Analysis.</em> New York, NY: W.H. Freeman and Company.</li>
</ol>

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

Concentrações de ácidos e bases

Acids and Bases
An Arrhenius acid produces hydrogen ions when it is dissolved in water:
HA + H2O &#8594; H+(aq) + A-(aq)
Here, HA is the non-dissociated ...

Esterification

<h4><strong>Esters</strong></h4>
<p>The structure of an ester is a carbonyl with an alkyl or aryl group (R) on one side, and an oxygen bound to another alkyl or aryl group (R&#8217;) on the other side, represented by the general formula: RCOOR&#8217;. Esters can be commonly derived by an esterification reaction between a carboxylic acid and an alcohol. The hydroxyl group of the carboxylic acid is replaced by an alkyl or aryl group. Simple esters, which have low molecular weight and small R and R&#8217; functional groups, have smells and flavors of fruits and flowers.</p>
<p>The simplest carboxylic acid is formic acid (HCOOH), with the simplest alcohol being methanol. Thus, the simplest ester formed from the esterification of these two molecules is methyl formate (HCOOCH<sub>3</sub>). Methyl formate is considered a derivative of formic acid because the OH group is replaced by the OCH<sub>3</sub> group. The structural differences between methyl formate and formic acid dictate the difference in their properties. This is because the properties of the molecule depend heavily on the R and R&#8217; groups.</p>
<p>Esters that are found in fruits and flowers are known as simple esters due to the low complexity of their structure in comparison to other esters. The alkyl and aryl groups found on these esters greatly influences the fragrance they emit; thus, the addition of a carbon or a different functional group can impact the scent produced by the ester. For example, propyl acetate (C<sub>5</sub>H<sub>10</sub>O<sub>2</sub>) has the scent of pears. However, butyl acetate (C<sub>6</sub>H<sub>12</sub>O<sub>2</sub>), which has only one more carbon in its chain, smells like apples.</p>
<p>Long-chain esters, meaning those with long alkyl chains, are called fatty acids. Fatty acids are important biological compounds that are essential for certain biological processes. Fatty acids are also a biological form of energy storage. When three fatty acids react with glycerol, they form a triglyceride &#8212; the most common form of fatty acids. Triglycerides have a very high molecular weight and can be highly branched. Their function and properties are dependent on the length and types of alkyl chains incorporated into their structure. One of the most common industrial uses for triglycerides is in the production of soaps and detergents.</p>
<h4><strong>Esterification Reaction</strong></h4>
<p>Esters are formed from an esterification reaction, with simple esters being formed through Fisher esterification. This reaction converts a carboxylic acid and alcohol into an ester with water as a by-product. Fisher esterification is a reversible reaction that proceeds very slowly. An acid catalyst, typically in the form of sulfuric acid, is added to increase the rate of the reaction while also acting as a dehydrating agent.</p>
<p>The mechanism of action for a Fisher esterification begins by the carbonyl oxygen attacking the sulfuric acid and deprotonating its OH group. This leads to a positively charged carbonyl oxygen. To better stabilize the positive charge, one electron pair of the carbonyl double bond is pushed to the carbonyl oxygen while simultaneously creating an electron-poor carbon center. Next, the hydroxy group of the alcohol acts as a nucleophile and attacks the electron-poor carbon &#8212; the electrophile &#8212; forming an intermediate. Then, the positively-charged hydroxyl group from the alcohol is deprotonated by the hydroxyl group of the carboxylic acid, stabilizing the oxygen atom of the alcohol. In the last step, the carbonyl oxygen is deprotonated by the conjugated base, and the free electron pair is moved towards the carbonyl center while the protonated carboxyl group leaves as a water molecule.</p>
<p>Since the esterification reaction is reversible, a 1:1 mixture of the carboxylic acid and the alcohol will reach equilibrium with about 70% yield of the ester. However, as with any equilibrium process, the reaction can be driven in one direction by changing the concentration, pressure, temperature, or volume of the reagents. This change causes the reaction to adjust to a new equilibrium to counteract the change, which is the basis of Le Chatelier&#8217;s principle.</p>
<p>When the concentration of one of the reactants is increased, the equilibrium shifts in the direction that will decrease its concentration. Thus, by increasing the concentration of one reactant, the equilibrium produces more of the product, thereby resulting in a higher yield of the ester product.</p>
<p>One common method utilized in organic chemistry labs is to provide an excess of one of the reagents, typically the alcohol. As mentioned earlier, Fisher esterification can utilize sulfuric acid as a catalyst. However, one of the other functions of sulfuric acid is to act as a dehydrating agent, sequestering water molecules away from the reaction. Applying Le Chatelier&#8217;s principle demonstrates that as one of the products is removed &#8212; in this case, water &#8212; the reaction is pushed to making more product. This increases the yield of the ester formed.</p>
<h4><strong>Reference</strong></h4>
<ol>
	<li style="font-size:17px">Streitwieser, A., Heathcock, C.H., Kosower, E.M. (1998). <em>Introduction to Organic Chemistry. </em>Upper Saddle River, NJ: Prentice Hall.</li>
</ol>

Esterification: Synthesis of Ester by Fischer Esterification - Concept

Esterificação

Esters
The structure of an ester is a carbonyl with an alkyl or aryl group (R) on one side, and an oxygen bound to another alkyl or aryl group (R&#8217;) ...

Enzyme Activity

<h4>Biological Catalysts</h4>

<p>All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require an input of energy to start, which is called the activation energy. Catalysts are chemicals that lower the activation energy. Even though catalysts facilitate a chemical reaction, they are not consumed by it. This means a catalyst can facilitate a specific chemical reaction over and over in succession, increasing the rate of that reaction.</p>

<p>Enzymes are biological catalysts that increase the speed and efficiency of biochemical reactions. Most enzymes are proteins but certain ribonucleic acid molecules also have catalytic properties. Each type of enzyme can only bind to one or a few specific substrates, therefore, there is a vast diversity of enzymes, all with distinct functions. The specific reaction of an enzyme depends on its active sites, which has shapes that closely matches the shape of its substrates. Upon substrate binding, the enzyme changes shape slightly in a way that allows the chemical reaction to happen more readily, thus reducing the activation energy of the reaction. Once the reaction takes place, the enzyme releases the products and returns to its original shape, which allows it to repeat the process again.</p>

<h4>Catabolytic versus Anabolic Enzymes</h4>

<p>Most enzymatic reactions can be categorized as either catabolic and anabolic, which have opposite functions. Enzymes that mediate catabolic reactions break down larger substances into multiple products. A well-known catabolic enzyme is lactase, which splits the disaccharide lactose in dairy products into monosaccharides galactose and glucose. Humans have large amounts of lactase in their small intestine after birth, however many individuals lose more than 90% of their lactase by early childhood<sup>1</sup>. This reduction diminishes the ability to digest lactose in an age-dependent manner, which is known as lactose intolerance<sup>2</sup>.</p>

<p>Anabolic enzymes combine multiple substances into a single product. For example, the enzyme DNA polymerase synthesizes DNA molecules by joining nucleotides together. Similarly, the enzyme glycogen synthase connects glucose molecules to generate glycogen. Insulin regulates the activity of glycogen synthase, therefore diabetic patients with low to no insulin levels cannot synthesize glycogen without insulin treatment<sup>3</sup>.</p>

<h4>Environmental Factors</h4>

<p>The shape and charge of the active site of an enzyme is tailored to the specific chemical reaction it catalyzes, therefore the enzyme activity is influenced by factors that affect its active site. Hence, enzymes require specific conditions to work at optimal levels and depending on their function and location, these conditions can vary greatly. These factors include pH and temperature. For example, active sites of enzymes often contain acidic or basic amino acids. Deviations from the optimum pH alter the charges in the active site, making it difficult for the substrates to interact with the enzyme. This property has been used for centuries to preserve food by pickling, which reduces the pH of the pickled food item to a level that effectively inhibits microbial enzymatic activity. Similarly, each enzyme works at an optimal temperature range. If the temperature shifts below or above this range, it alters the shape of the active site, preventing it from effectively interacting with its substrates. In most cases, returning to the optimal temperature restores the enzyme&#8217;s original shape and function, however, temperatures that are too high can denature or irreversibly damage the enzyme structure. Again, these properties are useful for food safety; we cook meat thoroughly to kill harmful bacteria by denaturing their enzymes and refrigerate most food items to slow down enzymatic activity of microorganisms.</p>

<p>Extremophiles, or organisms found in extreme conditions that are lethal to most forms of life, have enzymes capable of functioning at those extremes. For instance, <em>Thermus aquaticus</em> is a bacterium found in hot springs and hydrothermal vents reaching temperatures at which most proteins would denature. Taq polymerase is a thermostable DNA polymerase found in <em>T. aquaticus</em>, which has allowed the development of polymerase chain reaction (PCR), a widely-used laboratory method for rapid DNA replication. PCR requires high temperatures to separate the strands of the DNA double helix to be replicated. Taq polymerase remains stable at these high temperatures while most enzymes would be degraded. PCR cycles between high temperatures that break the DNA into single strands and lower temperatures that allow the polymerase to build new double stranded DNA.</p>

<h4>Molecular Factors that Influence Enzyme Activity</h4>

<p>In addition to specific pH and temperature ranges, some enzymes function better in the presence of other molecules or ions. Coenzymes are biomolecules that bind to the active site of the enzyme and participate in catalysis without getting consumed by the reaction. Coenzymes often transport electrons, specific atoms and functional groups, and thus act as intermediary carriers. B vitamins act as coenzymes for numerous biochemical reactions. For instance, coenzyme A (coA), which is the active form of pantothenic acid, is required by 4% of all mammalian enzymes<sup>4</sup>. Cofactors are often classified as inorganic substances, such as ions, that are required for the enzymatic function. For example, all DNA polymerase enzymes require Mg<sup>2+</sup> or Mn<sup>2+</sup> for DNA replication as well as excision of incorrectly incorporated nucleotides<sup>5</sup>.</p>

<p>There are also other molecules that are not necessary for an enzyme to function but are capable of altering the enzymatic activity. Substances that increase the enzyme activity are called activators, whereas others that decrease the enzyme activity are called inhibitors. These molecules bind to enzymes either at the active site or other locations on the enzyme known as allosteric sites. Sometimes these molecules can bind to the exact location in the active site as the substrate, and thus compete with the substrate for the enzyme binding. Others, especially the substances that bind to allosteric sites, can alter enzyme function without blocking the substrate binding to the active site, and are thus non-competitive. Moreover, binding of inhibitors can be reversible or irreversible. When the interaction is reversable, the enzyme can resume its function once the inhibitor detaches. However, when the binding is irreversible, the inhibitor never detaches from the enzyme and the enzyme becomes irreparably damaged and needs to be replaced. Nevertheless, enzyme inhibitors are useful as well. For instance, inhibitors have been utilized as antibiotics to inhibit bacterial growth, and as chemotherapeutics to inhibit the uncontrolled division of cancer cells<sup>6</sup>.</p>

<h4>Measuring Enzyme Function</h4>

<p>One method used by scientists to study the effects of different conditions on enzyme function is to quantify the reaction rate, which is how fast the enzyme is catalyzing the reaction. Therefore, a higher reaction rate indicates that the enzyme is catalyzing reactions more efficiently, whereas a lower rate means that it is working less efficiently. To determine the reaction rate, the reaction velocity is calculated by measuring the amount of product made per unit time. This is done by measuring the product concentration starting right at the beginning of the reaction when the product concentration is increasing linearly, until the enzyme is completely saturated with the substrate and the catalysis reaches a steady level. Once the baseline reaction rate is determined, it can be used as a reference point, or control, when comparing between a variety of treatments. We can assess reaction rates as relative or absolute measures. A relative reaction rate is a comparative measure that shows differences between two or more conditions, such as treatments in a single experiment, but does not give a value for what is being measured. An absolute reaction rate relies on a quantitative measure of the progress of the reaction. An easy way to understand the difference between relative and absolute reaction rates is to consider measuring the speed of a car in a race. A relative method would compare by assessing the ranking of cars at the finish line, whereas the absolute rate would assess and compare the speed of individual cars in miles per hour.</p>

<p>Since enzymes are so widely used, researchers have begun looking for ways to create and modify enzymes to suit specific needs, known as protein engineering. Recombinant DNA technology allows amino acid sequences to be changed in order to alter an enzyme&#8217;s shape or active site affinity. This has been used to create highly sensitive diagnostic tests for various conditions, as well as for bioremediation efforts and waste management tools<sup>7</sup>.</p>

<h4>References</h4>

<ol class="references">
	<li><strong>Swagerty DL Jr, Walling AD, Klein RM.</strong> Lactose intolerance. <em>Am Fam Physician. </em>2002, Vol. 65, 9: 1845-50.</li>
	<li><strong>Lomer MC, Parkes GC, Sanderson JD.</strong> Review article: lactose intolerance in clinical practice--myths and realities. <em>Aliment Pharmacol Ther. </em>2008, Vol. 27, 2: 93-103.</li>
	<li><strong>AR, Saltiel.</strong> New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. <em>Cell. </em>2001, Vol. 104, 4: 517-29.</li>
	<li><strong>DO, Kennedy.</strong> B Vitamins and the Brain: Mechanisms, Dose and Efficacy--A Review. <em>Nutrients. </em>2016, Vol. 8, 2: 68 doi:10.3390/nu8020068.</li>
	<li><strong>Vashishtha AK, Wang J, Konigsberg WH.</strong> Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases. <em>J Biol Chem. </em>2016, Vol. 291, 40: 20869-20875.</li>
	<li><strong>Watson ID, Stewart MJ, Platt DJ.</strong> Clinical pharmacokinetics of enzyme inhibitors in antimicrobial chemotherapy. <em>Clin Pharmacokinet. </em>1988, Vol. 15, 3: 133-64.</li>
	<li><strong>Ahuja SK, Ferreira, GM, Moreira AR.</strong> Utilization of Enzymes for Environmental Applications. <em>Crit Rev Biotech. </em>2004, Vols. 2-3, 125-54.</li>
</ol>


Enzyme Activity: Effect of pH and Temperature on Peroxidase Activity - Concept

Atividade enzimática

Biological Catalysts

All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require ...

Biologia

Processos Celulares

Hydrolysis of an Ester

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you get started, put on a lab coat, safety glasses, and nitrile gloves. Ensure that the lab is equipped with enough lab coats, splash goggles, and gloves for all of the students.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 1 L of 3 M NaOH. Measure 120 g of NaOH pellets, and transfer the pellets to a 1-L volumetric flask. Add deionized water to the 1-L line. Place a stir bar in the flask and mix the solution until the pellets are fully dissolved.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Once the solution is homogeneous, transfer the solution to a glass bottle, cap it, and label it. Place the bottle in a central hood so that students have access to it.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Prepare 1 L of 1 M CaCl<sub>2</sub> solution. Weigh 110.98 g of CaCl<sub>2</sub>, and transfer it to a 1-L volumetric flask. Fill the flask to the marked line with deionized water, add a stir bar, and mix the solution on a stir plate until the CaCl<sub>2</sub> is fully dissolved.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Once the solution is homogeneous, transfer it to a clean glass bottle, cap it, and label it. Store the solution in a central hood.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Prepare at least 1 L of 3 M HCl, which will be used to neutralize any excess sodium hydroxide at the end of the experiment.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Obtain 5 silicone soap molds and leave them in a common area. Obtain at least five different fragrance oils and five powder soap dyes in a variety of colors, and set them near the molds.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place a container of commercial liquid soap in a common area. Set coconut oil next to the balance making sure there are clean spatulas and weighing boats.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Package of pH paper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;pH meter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL plastic squeeze bottle of DI water</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;50-mL volumetric flasks</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;100-mL glass graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;6&#160;&#160;&#160;&#160;50-mL glass graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Glass vials</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small spoon</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small spatula</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

Hydrolysis of an Ester: Preparation of Soap - Prep

Hidrólise de um éster

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

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

Lab Manual Sub Articles

Identification of Unknown Aldehydes and Ketones

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Test Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you start, put on a lab coat, splash-proof safety glasses, and nitrile gloves. <strong>Note: </strong>DNPH is a shock explosive, so handle it gently with low friction tools. Phosphoric acid is also toxic and corrosive.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 100 mL of a 0.1 M solution of DNPH in a 50/50 mixture of 85% phosphoric acid and ethanol. Add a medium stir bar to a 250-mL beaker. In a fume hood, measure 3 g of DNPH and gently pour it into the beaker.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 50 mL of 85% by weight aqueous phosphoric acid and slowly add it to the beaker. Place the mixture on a stir plate and gently stir it until the DNPH dissolves.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Measure 50 mL of ethanol and pour it into the beaker. Once the solution looks homogeneous, turn off the stir motor and retrieve the stir bar.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Vacuum filter the solution to remove residual solids before transferring it to a 100-mL container. Cap the container, label it &#8216;DNPH solution&#8217;, and place it in a hood for the test reagents.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Prepare 200 mL of iodoform reagent. Measure 50 g of potassium iodide into a clean 250-mL beaker. Add 200 mL of deionized water and stir the mixture on a stir plate until the potassium iodide has completely dissolved.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure 25 g of iodine and add it to the stirring solution. Once the iodine has dissolved, turn off the stir motor. Transfer the solution to a labeled 200-mL container and cap it. Place the iodoform reagent in the test solution hood.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Make 22 mL of Tollens&#8217; reagent. Measure 0.6115 g of silver nitrate and pour it into a 50-mL beaker. <strong>Note: </strong>Prepare this solution right before the lab because explosive silver nitride will start to form as the reagent decomposes.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Add 12 mL of deionized water and stir until the silver nitrate has dissolved completely to form a 0.3 M silver nitrate solution.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure 6 mL of 3 M NaOH and add it to the stirring silver nitrate solution. Brown silver oxide will precipitate from the solution.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Measure 4 mL of 2 M aqueous ammonia. Use a Pasteur pipette to slowly add it to the stirring solution.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once you have added all 4 mL of ammonia, remove the stir bar and vacuum filter the solution. Pour the filtrate into a 25-mL brown glass bottle.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Wrap the bottle in foil to shield it from light and label it &#8216;Tollens&#39; Reagent&#8217;. Put the wrapped bottle with the other test solutions.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Make sure that the sinks have paper towels and test tube brushes.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Set up appropriate waste containers in a waste collection hood and place glass waste nearby.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Fill a wash bottle with acetone and place it in the waste hood so that students can rinse strong-smelling chemicals out of their Pasteur pipettes before disposing of them.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">In a fume hood, pour about 5 mL of butanone in a labeled vial for each lab group.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Prepare and label vials containing 5 mL of benzaldehyde in the same way. These are the known compounds that every student group will need.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">The unknown compounds are acetophenone, butanol, 3-methyl-2-butanone, pentanol, 2-pentanone, and 3-pentanone. For each compound, prepare two vials containing 5 mL of that compound and label them with a code name. Place the known and unknown vials in a central area.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab stand</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small 3-prong clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Vacuum line</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Test tube rack</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Test tube tongs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;4&#160;&#160;&#160;&#160;250-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;150-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;5&#160;&#160;&#160;&#160;50-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;10-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;5&#160;&#160;&#160;&#160;5-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL filter flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;B&#252;chner funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Filter adapter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;4&#160;&#160;&#160;&#160;Small watch glasses</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Thermometer with beaker clip</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;Pipette bulbs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Package of pH paper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of lab tape and marking pen</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL bottle of DI water</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Put Pasteur pipettes, filter papers, and small test tubes in a central area.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place 3 M NaOH and 1,2-dimethoxyethane in a hood reserved for solvents.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Set up the melting point analyzer and ensure that there are enough capillaries.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">At the end of the lab, promptly dilute any unused Tollens&#39; reagent with at least 20 mL of water and flush it down the drain with copious tap water. <strong>Note: </strong>If necessary, use concentrated hydrochloric acid to precipitate the silver from the Tollens&#39; reagent waste first.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Clean silver-coated test tubes with concentrated nitric acid in a fume hood. Neutralize and dispose of the silver waste according to your lab&#39;s standard procedures.</div>
		</li>
	</ul>
	</li>
</ol>

Test for Aldehydes and Ketones: DNPH, Tollen's Test, and Iodoform Test - Prep

Identificação de aldeídos e cetonas desconhecidos

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

	Preparation of Test SolutionsExpand
	Here, we show the laboratory preparation ...

Proper Lab Notebook Keeping

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Put on a lab coat, splash-proof safety glasses, and nitrile gloves. You&#39;ll work in a fume hood because these acids are toxic and corrosive.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Ensure that the lab has containers for concentrated acid waste, aqueous acid waste, and glass waste.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Prepare a bottle of concentrated sulfuric acid for each student group. Measure 10 mL of 98% w/w sulfuric acid with a graduated cylinder and pour it into 20-mL bottles using a funnel. Tightly cap the bottles and label them with the solution name and concentration.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Prepare a bottle of nitric acid for each student group. Measure 10 mL of 70% w/w nitric acid with a clean graduated cylinder and pour it into 20-mL bottles via a clean funnel. Cap the bottles and label them with the name and concentration.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Set one bottle of nitric acid and one bottle of sulfuric acid at each hood.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Place a bottle of methyl benzoate in a common hood.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Put Pasteur pipettes and circular filter papers on a central bench.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Confirm that the balance area has enough weighing boats, spatulas, and lab wipes for the class.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;Lab stands</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3&#160;&#160;&#160;&#160;Small 2-prong clamps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;100-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;250-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3&#160;&#160;&#160;&#160;10-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;50-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;25-mL Erlenmeyer flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;250-mL filter flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;20-mL test tube</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Large B&#252;chner funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Rubber filter adapter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Thermometer</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;Pipette bulbs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Roll of lab tape</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Marker/pen</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3&#160;&#160;&#160;&#160;Small beakers</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

Manutenção adequada do caderno de laboratório

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

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

Synthesis of Luminol

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you get started, put on a lab coat, splash-proof safety glasses, and nitrile gloves. The chemicals that you will use are hazardous, so both solutions must be prepared and handled in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 10 mL of a 10% by volume solution of hydrazine in water. Measure 8.4 mL of deionized water and add it to a 30-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 1.6 mL of hydrazine hydrate and pour it into the beaker for a total volume of 10 mL. Stir the hydrazine solution with a glass rod until it is well mixed. <strong>Note:</strong> Hydrazine is flammable, toxic, and explosive.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Pour the solution into a 20-mL glass bottle. Cap it and label it with the solution&#39;s name and concentration. Store the hydrazine hydrate and the hydrazine solution in a cabinet for flammables.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Prepare 20 mL of 2.8 to 3 M aqueous NaOH. <strong>Note:</strong> NaOH is highly corrosive. Avoid skin contact.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Measure 2.2 to 2.4 g of solid NaOH and add it to a 50-mL beaker. Add 20 mL of deionized water and stir the mixture on a stir plate until the NaOH has completely dissolved.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Pour the solution into a 20-mL glass bottle and close it with a lid. Label the bottle with the solution&#39;s name and approximate concentration. Store the NaOH pellets and solution in the appropriate cabinets.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Lastly, clean your glassware following your usual procedures.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Before you set up the main lab, reserve a separate room that you can make as dark as possible.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Prepare a container for fluorescein waste and ensure that a glass waste container is available.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Set out a 10% sodium hypochlorite (bleach) solution and a 1 M sodium carbonate solution in the waste hood.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Fill a wash bottle with deionized water and place it in the same hood. Confirm that there are paper towels and a test tube brush by each sink in the main lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Bring containers of 3-nitrophthalic acid, anhydrous sodium dithionite, solid potassium hydroxide, and fluorescein to the balance area.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Confirm that there are enough laboratory wipes, spatulas, and weighing boats for the class.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set triethylene glycol, the hydrazine solution, the NaOH solution, glacial acetic acid, and dimethyl sulfoxide in a central hood. Place a 1-mL graduated pipette and a pipette controller designated for hydrazine in the same hood.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Put Pasteur pipettes, filter papers, scissors, and plastic paraffin film in a central area.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stirring hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Bunsen burner with tubing</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Lab stands</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;Clamps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Pair of flask tongs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;4&#160;&#160;&#160;&#160;10-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;25-mL Erlenmeyer flasks</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL filter flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;250-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;25-mL test tubes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;B&#252;chner funnels</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Long glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Medium stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Thermometers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Spatulas</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;6&#160;&#160;&#160;&#160;Boiling chips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Medium filter adapter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Test tube stopper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small flask stopper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Pair of tweezers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Fire striker or lighter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of laboratory tape or labels</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Pipette bulbs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Pen</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

Synthesis of Luminol and Chemiluminescence - Prep

Síntese de Luminol

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

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

Beer's Law

Beer-Lambert Law and Equilibrium Constant Determination - Procedure

Lei da Cerveja

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

	Lab Setup for Beer&#8217;s LawExpand
	In this lab, you will combine aqueous solutions of FeCl3 ...