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

La cromatografía en columna es una técnica que utiliza una columna empaquetada para separar compuestos en función de sus interacciones con una fase estacionaria, que suele ser en forma de perlas microscópicas. Los espacios entre las cuentas se rellenan con disolvente. La apertura de la columna permite que el solvente fluya a través de la fase estacionaria. Cuando se aplica una mezcla a la parte superior de la columna empaquetada seguida de una mayor cantidad de solvente, la mezcla pasa a la fase móvil y fluye a través de la fase estacionaria.
Cada componente de la mezcla interactúa con la fase estacionaria de una manera diferente. Algunos componentes tienen interacciones débiles con la fase estacionaria y, por lo tanto, se mueven rápidamente a través de la columna, mientras que otros tienen interacciones fuertes con la fase estacionaria y, por lo tanto, se mueven más lentamente. Esto separa los diferentes compuestos en bandas, que se recogen en pequeñas fracciones. Esto permite la purificación de cada compuesto.
Entonces, ¿qué tipo de propiedades se pueden utilizar para separar mezclas? Una de las propiedades más utilizadas es la polaridad. Para ello, el gel de sílice, que es una forma de dióxido de silicio, se utiliza a menudo como fase estacionaria. El gel de sílice interactúa con los compuestos mediante interacciones dipolo-dipolo y enlaces de hidrógeno a través de los grupos -OH que se forman en su superficie. Por lo tanto, los compuestos polares interactuarán fuertemente con la fase estacionaria, mientras que los compuestos no polares interactuarán débilmente.
Otras propiedades que se pueden aprovechar para la separación incluyen el tamaño, la carga y la hidrofobicidad. Una forma común de cargar la fase estacionaria en la columna es como una suspensión de la fase estacionaria y solvente. Luego, la fase estacionaria se empaqueta haciendo fluir más solvente a través de la columna para compactar la suspensión. Es esencial que la fase estacionaria se llene de manera uniforme, sin burbujas de aire, canales vacíos o parches secos. Estos pueden interrumpir el flujo y causar la mezcla de bandas.
Al elegir una columna, el diámetro se basa en el volumen de la muestra que se va a separar. La muestra debe cubrir la parte superior de la columna con una capa delgada y uniforme. Una capa de muestra más gruesa conduce a bandas más anchas. La longitud de la columna depende de lo bien que se separen los compuestos en la fase estacionaria. Los compuestos con afinidades similares por la fase estacionaria requieren una columna larga para una separación adecuada. Sin embargo, una mezcla de compuestos con afinidades muy diferentes para la fase estacionaria se puede separar en una columna más corta.
En este laboratorio, explorará la cromatografía en columna empaquetando y preparando primero una columna de gel de sílice. Luego, usará su columna para separar los componentes coloreados en colorante alimentario verde.

cromatografía en columna

Cromatografía en columna
Existen múltiples técnicas para purificar y separar compuestos en un laboratorio de química orgánica. Una de las técnicas de separación más fiables para las separaciones a microescala, en las que se dispone de menos de 10 gramos del compuesto para su análisis, es la cromatografía en columna. Esta técnica separa los compuestos de una mezcla en función de sus propiedades físicas, como su solubilidad, polaridad, hidrofobicidad, tamaño y carga.
<h4>Conceptos básicos de cromatografía</h4>
Los componentes principales de cualquier sistema de cromatografía son la fase estacionaria, la fase móvil y la mezcla de muestras a analizar. En la cromatografía en columna, la fase estacionaria suele ser de perlas a microescala, que se empaquetan uniformemente en una columna vertical. A la parte superior de la columna se añade un flujo continuo de la fase móvil, también conocida como disolvente, que fluye a través de la fase estacionaria por gravedad o a un caudal controlado mediante una bomba.
La muestra se disuelve en una pequeña cantidad de disolvente y luego se agrega a la parte superior de la columna. Se añade más disolvente para forzar el flujo de la muestra a través de la fase estacionaria. Los compuestos de la mezcla se dividen entre la fase móvil y la fase estacionaria en función de sus diferentes propiedades y forman bandas discretas.
Los compuestos con interacciones fuertes con la fase estacionaria se moverán más lentamente que los compuestos con interacciones débiles con la fase estacionaria. Por lo tanto, los compuestos con interacciones débiles saldrán de la columna, o eluirán, antes que aquellos con interacciones fuertes. La fuerza de la interacción de un compuesto con la fase estacionaria está definida por el factor de retardo (Rf), que es la relación entre la distancia recorrida por un componente y la recorrida por la fase móvil. El factor de retardo se determina realizando primero una cromatografía en capa fina.
<img src="/CDNSource/lm/labs/63/63_Concepts_1.jpg">
Un valor alto del factor de retardo indica que la muestra interactúa fuertemente con la fase estacionaria y tarda mucho tiempo en eluir. Un valor bajo del factor de retardo indica que la muestra no interactúa con la fase estacionaria muy fuertemente y eluye más rápidamente. Dado que los compuestos se dividen en bandas individuales y eluyen de la columna en diferentes momentos debido a sus diferentes valores de Rf, se pueden aislar individualmente recogiendo el disolvente de debajo de la columna en fracciones.
<h4>Consideraciones sobre la cromatografía en columna</h4>
Una columna de cromatografía es un tubo de vidrio o plástico orientado verticalmente. El tamaño de la columna puede variar desde unos pocos centímetros (como una pipeta Pasteur) hasta metros (como en el caso de las columnas industriales). El diámetro de la columna depende de la cantidad de muestra a separar, mientras que la longitud de la columna depende de la dificultad de separación. La partición más eficiente en bandas discretas requiere capas de muestra delgadas; Por lo tanto, determinar el diámetro adecuado es un paso vital. La carga de demasiado volumen de muestra en relación con el diámetro de la columna creará bandas más anchas y menos definidas que el mismo volumen de muestra en una columna con un diámetro mayor.
A la hora de elegir la longitud de la columna, hay que tener en cuenta los valores Rf de los componentes. Los componentes con factores de retardo similares pueden ser difíciles de separar y requieren una columna más larga para resolver las bandas individuales. Es posible que los compuestos con factores de retardo muy diferentes no necesiten una columna larga, ya que deberían dividirse fácilmente. A la hora de seleccionar la fase estacionaria, es importante elegir una que dé lugar a diferentes factores de retardo para cada componente.
La preparación de la columna es la parte más crítica del funcionamiento de una columna de cromatografía. La masa de la fase estacionaria empaquetada en la columna debe ser al menos 20 veces mayor que la de la muestra. Si la columna no tiene un disco poroso en la parte inferior, debe empacarse con algodón y una fina capa de arena. Esto evita la pérdida de la fase estacionaria a través de la salida de la columna.
Hay dos métodos para empacar la columna: el método de empaque en seco y el método de lodo. En el método seco, la fase estacionaria se transfiere a la columna en su forma de polvo. A continuación, la columna se lava varias veces con la fase móvil/disolvente para garantizar que el disolvente penetre en todas las partes de la columna de gel de sílice. Este método, si no se hace correctamente, puede aumentar la probabilidad de que se formen parches secos, canales y burbujas de aire en la columna. Estos defectos afectarán a la capacidad de la columna para dividir los componentes de una mezcla.
El método de lechada proporciona una columna empaquetada más uniforme que no contiene burbujas de aire, áreas secas ni canales. Para este método, la fase estacionaria se mezcla con un solvente para formar una suspensión consistente. A continuación, la lechada se transfiere a la columna mientras se golpea en los lados para eliminar las burbujas de aire formadas. La llave de paso de la columna, si la hay, debe estar abierta durante este paso para permitir que el solvente drene.
<h4>Cromatografía en gel de sílice</h4>
Se explotan muchas propiedades diferentes para separar una mezcla de compuestos, como el tamaño, la carga y la hidrofobicidad. Una propiedad utilizada para separar compuestos en química orgánica es la polaridad. Para ello, el gel de sílice y el gel de alúmina son las fases estacionarias más utilizadas. El gel de sílice es extremadamente polar y forma fuertes interacciones dipolo-dipolo con los compuestos polares. Además, el gel de sílice puede formar enlaces de hidrógeno con el analito debido a la presencia de grupos -OH en su superficie.
Los componentes de la mezcla que son más polares se unen fuertemente al gel de sílice y viajan lentamente a través de la columna, mientras que los componentes no polares son más solubles en la fase móvil y viajan rápidamente a través de la columna. Por lo tanto, es esencial que los componentes de la mezcla tengan diferentes polaridades. Los componentes que interactúan fuertemente con la fase estacionaria se eluyen haciendo fluir una fase móvil polar a través de la columna.
<h4>Referencias</h4>
<ol>
	<li style="font-size:17px">Shuler, M.L., Kargi, K., DeLisa, M. (2017). Ingeniería de Bioprocesos, Conceptos Básicos. Upper Saddle River, Nueva Jersey: Prentice Hall.</li>
	<li style="font-size:17px">Harris, D.C. (2015). Análisis químico cuantitativo. Nueva York, NY: W.H. Freeman and Company.</li>
</ol>

Cromatografía en columna

Cromatografía en columna
Existen múltiples técnicas para purificar y separar compuestos en un laboratorio de química orgánica. Uno de los sistemas de separación ...

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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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