Name: separating mixtures by polarity
Uploaded: 2020-03-26T07:22:28.000Z
Duration: 3 min 7 s
Channel: JoVE

A cromatografia em coluna é uma técnica que usa uma coluna compactada para separar compostos com base em suas interações com uma fase estacionária, que geralmente está na forma de esferas microscópicas. Os espaços entre os grânulos são preenchidos com solvente. A abertura da coluna permite que o solvente flua através da fase estacionária. Quando uma mistura é aplicada no topo da coluna compactada seguida por mais solvente, a mistura se move para a fase móvel e flui através da fase estacionária.
Cada componente da mistura interage com a fase estacionária de uma maneira diferente. Alguns componentes têm interações fracas com a fase estacionária e, portanto, movem-se rapidamente pela coluna, enquanto outros têm fortes interações com a fase estacionária e, portanto, movem-se mais lentamente. Isso separa os diferentes compostos em bandas, que são coletadas em pequenas frações. Isso permite a purificação de cada composto.
Então, que tipo de propriedades podem ser usadas para separar misturas? Uma das propriedades mais comumente usadas é a polaridade. Para isso, o gel de sílica, que é uma forma de dióxido de silício, é frequentemente usado como fase estacionária. O gel de sílica interage com compostos por interações dipolo-dipolo e ligações de hidrogênio através dos grupos -OH que se formam em sua superfície. Assim, os compostos polares interagirão fortemente com a fase estacionária, enquanto os compostos apolares interagirão fracamente.
Outras propriedades que podem ser exploradas para separação incluem tamanho, carga e hidrofobicidade. Uma maneira comum de carregar a fase estacionária na coluna é como uma pasta da fase estacionária e do solvente. Em seguida, a fase estacionária é embalada fluindo mais solvente através da coluna para compactar a pasta. É essencial que a fase estacionária seja embalada uniformemente, sem bolhas de ar, canais vazios ou manchas secas. Isso pode interromper o fluxo e causar a mistura de bandas.
Ao escolher uma coluna, o diâmetro é baseado no volume da amostra a ser separada. A amostra deve cobrir o topo da coluna em uma camada fina e uniforme. Uma camada de amostra mais espessa leva a bandas mais largas. O comprimento da coluna depende de quão bem os compostos se separam na fase estacionária. Compostos com afinidades semelhantes para a fase estacionária requerem uma coluna longa para separação adequada. No entanto, uma mistura de compostos com afinidades muito diferentes para a fase estacionária pode ser separada em uma coluna mais curta.
Neste laboratório, você explorará a cromatografia em coluna primeiro empacotando e preparando uma coluna de sílica gel. Em seguida, você usará sua coluna para separar os componentes coloridos em corante alimentar verde.

cromatografia em coluna

Cromatografia em coluna
Existem várias técnicas para purificar e separar compostos em um laboratório de química orgânica. Uma das técnicas de separação mais confiáveis para separações em microescala, onde menos de 10 gramas do composto estão disponíveis para análise, é a cromatografia em coluna. Essa técnica separa os compostos em uma mistura com base em suas propriedades físicas, como solubilidade, polaridade, hidrofobicidade, tamanho e carga.
<h4>Noções básicas de cromatografia</h4>
Os principais componentes de qualquer sistema de cromatografia são a fase estacionária, a fase móvel e a mistura de amostras a ser analisada. Na cromatografia em coluna, a fase estacionária é tipicamente esferas de microescala, que são embaladas uniformemente em uma coluna vertical. Um fluxo contínuo da fase móvel, também conhecido como solvente, é adicionado ao topo da coluna, que flui através da fase estacionária por gravidade ou a uma taxa de fluxo controlada por uma bomba.
A amostra é dissolvida em uma pequena quantidade do solvente e depois adicionada ao topo da coluna. Mais solvente é adicionado para forçar a amostra a fluir através da fase estacionária. Os compostos na mistura dividem-se entre a fase móvel e a fase estacionária com base em suas propriedades diferentes e formam bandas discretas.
Compostos com fortes interações com a fase estacionária se moverão mais lentamente do que compostos com interações fracas com a fase estacionária. Assim, compostos com interações fracas sairão da coluna, ou eluirão, mais cedo do que aqueles com interações fortes. A força da interação de um composto com a fase estacionária é definida pelo fator de retardo (R f), que é a razão entre a distância percorrida por um componente e a percorrida pela fase móvel. O fator de retardo é determinado pela primeira execução de cromatografia em camada fina.
<img src="/CDNSource/lm/labs/63/63_Concepts_1.jpg">
Um alto valor de fator de retardo indica que a amostra interage fortemente com a fase estacionária e leva muito tempo para eluir. Um baixo valor de fator de retardo indica que a amostra não interage com a fase estacionária com muita força e elui mais rapidamente. Como os compostos se dividem em bandas individuais e eluem da coluna em momentos diferentes devido aos seus diferentes valores de Rf, eles podem ser isolados individualmente coletando o solvente sob a coluna em frações.
<h4>Considerações sobre cromatografia em coluna</h4>
Uma coluna de cromatografia é um tubo de vidro ou plástico orientado verticalmente. O tamanho da coluna pode variar de alguns centímetros (como uma pipeta Pasteur) a metros (como em colunas industriais). O diâmetro da coluna depende da quantidade de amostra a ser separada, enquanto o comprimento da coluna depende da dificuldade de separação. O particionamento mais eficiente em bandas discretas requer camadas de amostra finas; portanto, determinar o diâmetro apropriado é uma etapa vital. Carregar muito volume de amostra em relação ao diâmetro da coluna criará faixas mais largas e menos definidas do que o mesmo volume de amostra em uma coluna com um diâmetro maior.
Ao escolher o comprimento da coluna, os valores Rf dos componentes devem ser considerados. Componentes com fatores de retardo semelhantes podem ser difíceis de separar e exigir uma coluna mais longa para resolver as bandas individuais. Compostos com fatores de retardo muito diferentes podem não precisar de uma coluna longa, pois devem se particionar facilmente. Ao selecionar a fase estacionária, é importante escolher uma que resulte em diferentes fatores de retardo para cada componente.
Preparar a coluna é a parte mais crítica da execução de uma coluna de cromatografia. A massa da fase estacionária embalada na coluna deve ser pelo menos 20 vezes maior que a da amostra. Se a coluna não tiver um disco poroso na parte inferior, ela deve ser embalada com algodão e uma fina camada de areia. Isso evita a perda da fase estacionária pela saída da coluna.
Existem dois métodos para embalar a coluna: o método de embalagem a seco e o método de pasta. No método seco, a fase estacionária é transferida para a coluna em sua forma de pó. A coluna é então lavada várias vezes com a fase móvel/solvente para garantir que o solvente penetre em todas as partes da coluna de sílica gel. Este método, se não for feito corretamente, pode aumentar a probabilidade de manchas secas, canais e bolhas de ar se formarem na coluna. Esses defeitos afetarão a capacidade da coluna de particionar componentes de uma mistura.
O método de lama fornece uma coluna compactada mais uniforme que não contém bolhas de ar, áreas secas e canais. Para este método, a fase estacionária é misturada com um solvente para formar uma pasta consistente. A pasta é então transferida para a coluna enquanto bate nas laterais para se livrar de quaisquer bolhas de ar formadas. A torneira da coluna, se houver, deve estar aberta durante esta etapa para permitir que o solvente escorra.
<h4>Cromatografia de sílica gel</h4>
Muitas propriedades diferentes são exploradas para separar uma mistura de compostos, como tamanho, carga e hidrofobicidade. Uma propriedade usada para separar compostos em química orgânica é a polaridade. Para isso, o gel de sílica e o gel de alumina são as fases estacionárias mais comumente utilizadas. O gel de sílica é extremamente polar e forma fortes interações dipolo-dipolo com compostos polares. Além disso, o gel de sílica pode formar ligações de hidrogênio com o analito devido à presença de grupos -OH em sua superfície.
Os componentes da mistura mais polares ligam-se fortemente ao gel de sílica e viajam lentamente pela coluna, enquanto os componentes não polares são mais solúveis na fase móvel e viajam rapidamente pela coluna. Assim, é essencial que os componentes da mistura tenham polaridades diferentes. Os componentes que interagem fortemente com a fase estacionária são eluídos pelo fluxo de uma fase móvel polar através da coluna.
<h4>Referências</h4>
<ol>
	<li style="font-size:17px">Shuler, M.L., Kargi, K., DeLisa, M. (2017). Engenharia de Bioprocessos, Conceitos Básicos. Rio Saddle Superior, NJ: Prentice Hall.</li>
	<li style="font-size:17px">Harris, D.C. (2015). Análise Química Quantitativa. Nova York, NY: W.H. Freeman and Company.</li>
</ol>

Cromatografia em coluna

Cromatografia em coluna
Existem várias técnicas para purificar e separar compostos em um laboratório de química orgânica. Uma das separações mais confiáveis ...

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Column Chromatography

separating mixtures by polarity

Column chromatography separates compounds in an organic chemistry lab by using differences in physical properties. It is especially useful for microscale separations when less than 10 grams of material is available for analysis. The method can separate a mixture based on solubility, polarity, hydrophobicity, size, and charge.

Any chromatography system has three main parts: the stationary phase, the mobile phase, and the sample mixture. In column chromatography, the stationary phase is usually made of microscale beads packed into a vertical column. The mobile phase is a solvent that flows through the column by gravity or at a controlled rate with a pump. The sample is first dissolved in a small amount of solvent, added to the top of the column, and then pushed through with more solvent.

As the mixture moves through the column, its compounds partition between the two phases. They form separate bands because each compound interacts differently with the stationary phase. Compounds that interact strongly move more slowly. Compounds with weaker interactions elute, or leave the column, earlier. The retardation factor, or Rf, describes this interaction by comparing how far a component travels with how far the mobile phase travels. A high Rf means strong interaction and slow elution. A low Rf means weaker interaction and faster elution. The Rf is determined first with thin layer chromatography, and the separated compounds are collected in fractions as they leave the column.

The size and shape of the column affect how well the separation works. A chromatography column is a vertical glass or plastic tube that can range from a few centimeters, such as a Pasteur pipette, to meters in industrial systems. The column diameter depends on the amount of sample, and the length depends on how difficult the separation is. Thin sample layers give the best band separation, so using the right diameter is important. If too much sample is loaded for the column diameter, the bands become broader and less defined. When components have similar Rf values, a longer column may be needed to separate them. If their Rf values are very different, a shorter column may still work well. The stationary phase should also be chosen so the components have different Rf values.

Packing the column correctly is one of the most important steps. The stationary phase should weigh at least 20 times more than the sample. If there is no porous disk at the bottom, cotton and a thin layer of sand are used to stop the stationary phase from leaving the column. The column can be packed by a dry method or by a slurry method. In the dry method, the powder is added directly and then washed with solvent so the mobile phase can move through the silica gel. If this step is not done well, dry patches, channels, and air bubbles can form. These defects reduce how well the mixture separates. The slurry method mixes the stationary phase with solvent first, then transfers it into the column while tapping the sides to remove bubbles. If the column has a stopcock, it should be open so the solvent can drain.

Silica gel chromatography is a common way to separate compounds by polarity. Silica gel and alumina gel are the most used stationary phases for this purpose. Silica gel is very polar and can form dipole-dipole interactions and hydrogen bonds because of its -OH groups. Polar components bind more strongly to silica gel and move slowly through the column. Non-polar components stay more soluble in the mobile phase and move faster. Because of this, the mixture must contain components with different polarities. More polar compounds are eluted by using a polar mobile phase through the column.

Separating Mixtures by Polarity

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Separating Mixtures by Polarity

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