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

La cromatografia su colonna è una tecnica che utilizza una colonna impaccata per separare i composti in base alle loro interazioni con una fase stazionaria, che di solito è sotto forma di microsfere microscopiche. Gli spazi tra le perle sono riempiti con solvente. L'apertura della colonna consente al solvente di fluire attraverso la fase stazionaria. Quando una miscela viene applicata alla sommità della colonna impaccata seguita da una maggiore quantità di solvente, la miscela passa alla fase mobile e scorre attraverso la fase stazionaria.
Ogni componente della miscela interagisce con la fase stazionaria in modo diverso. Alcuni componenti hanno interazioni deboli con la fase stazionaria e quindi si muovono rapidamente attraverso la colonna, mentre altri hanno forti interazioni con la fase stazionaria e quindi si muovono più lentamente. Questo separa i diversi composti in bande, che vengono raccolte in piccole frazioni. Ciò consente la purificazione di ogni composto.
Quindi, che tipo di proprietà possono essere utilizzate per separare le miscele? Una delle proprietà più comunemente usate è la polarità. Per questo, il gel di silice, che è una forma di biossido di silicio, viene spesso utilizzato come fase stazionaria. Il gel di silice interagisce con i composti mediante interazioni dipolo-dipolo e legami idrogeno attraverso i gruppi -OH che si formano sulla sua superficie. Pertanto, i composti polari interagiranno fortemente con la fase stazionaria, mentre i composti non polari interagiranno debolmente.
Altre proprietà che possono essere sfruttate per la separazione includono le dimensioni, la carica e l'idrofobicità. Un modo comune per caricare la fase stazionaria nella colonna è come un impasto di fase stazionaria e solvente. Quindi, la fase stazionaria viene impacchettata facendo scorrere più solvente attraverso la colonna per compattare il liquame. È essenziale che la fase stazionaria sia impacchettata in modo uniforme senza bolle d'aria, canali vuoti o zone secche. Questi possono interrompere il flusso e causare la miscelazione delle bande.
Nella scelta di una colonna, il diametro si basa sul volume del campione da separare. Il campione dovrebbe coprire la parte superiore della colonna in uno strato sottile e uniforme. Uno strato di campione più spesso porta a bande più ampie. La lunghezza della colonna dipende da quanto bene i composti si separano nella fase stazionaria. I composti con affinità simili per la fase stazionaria richiedono una lunga colonna per un'adeguata separazione. Tuttavia, una miscela di composti con affinità molto diverse per la fase stazionaria può essere separata su una colonna più corta.
In questo laboratorio, esplorerai la cromatografia su colonna impacchettando e preparando prima una colonna di gel di silice. Quindi, utilizzerai la tua colonna per separare i componenti colorati nel colorante alimentare verde.

cromatografia su colonna

Cromatografia su colonna
Esistono diverse tecniche per purificare e separare i composti in un laboratorio di chimica organica. Una delle tecniche di separazione più affidabili per le separazioni su microscala, in cui sono disponibili meno di 10 grammi del composto per l'analisi, è la cromatografia su colonna. Questa tecnica separa i composti in una miscela in base alle loro proprietà fisiche, come la solubilità, la polarità, l'idrofobicità, le dimensioni e la carica.
<h4>Nozioni di base sulla cromatografia</h4>
I componenti principali di qualsiasi sistema cromatografico sono la fase stazionaria, la fase mobile e la miscela di campioni da analizzare. Nella cromatografia su colonna, la fase stazionaria è tipicamente costituita da microsfere su microscala, che vengono impacchettate uniformemente in una colonna verticale. Un flusso continuo della fase mobile, nota anche come solvente, viene aggiunto alla sommità della colonna, che scorre attraverso la fase stazionaria per gravità o a una portata controllata da una pompa.
Il campione viene disciolto in una piccola quantità di solvente e quindi aggiunto alla parte superiore della colonna. Viene aggiunto altro solvente per forzare il flusso del campione attraverso la fase stazionaria. I composti nella miscela si dividono tra la fase mobile e la fase stazionaria in base alle loro diverse proprietà e formano bande discrete.
I composti con forti interazioni con la fase stazionaria si muoveranno più lentamente dei composti con deboli interazioni con la fase stazionaria. Pertanto, i composti con interazioni deboli usciranno dalla colonna, o eluiranno, prima di quelli con interazioni forti. La forza dell'interazione di un composto con la fase stazionaria è definita dal fattore di ritardo (Rf), che è il rapporto tra la distanza percorsa da un componente e quella percorsa dalla fase mobile. Il fattore di ritardo viene determinato eseguendo prima la cromatografia su strato sottile.
<img src="/CDNSource/lm/labs/63/63_Concepts_1.jpg">
Un valore elevato del fattore di ritardo indica che il campione interagisce fortemente con la fase stazionaria e impiega molto tempo per eluire. Un valore basso del fattore di ritardo indica che il campione non interagisce molto fortemente con la fase stazionaria ed eluisce più rapidamente. Poiché i composti si dividono in bande individuali ed eluiscono dalla colonna in momenti diversi a causa dei loro diversi valori di Rf, possono essere isolati individualmente raccogliendo il solvente da sotto la colonna in frazioni.
<h4>Considerazioni sulla cromatografia su colonna</h4>
Una colonna cromatografica è un tubo di vetro o plastica orientato verticalmente. La dimensione della colonna può variare da pochi centimetri (come una pipetta Pasteur) a metri (come con le colonne industriali). Il diametro della colonna dipende dalla quantità di campione da separare, mentre la lunghezza della colonna dipende dalla difficoltà di separazione. Il partizionamento più efficiente in bande discrete richiede strati di campioni sottili; Pertanto, determinare il diametro appropriato è un passaggio fondamentale. Caricare troppo volume di campione rispetto al diametro della colonna creerà bande più larghe e meno definite rispetto allo stesso volume di campione in una colonna con un diametro maggiore.
Nella scelta della lunghezza della colonna, è necessario considerare i valori Rf dei componenti. I componenti con fattori di ritardo simili possono essere difficili da separare e richiedere una colonna più lunga per risolvere le singole bande. I composti con fattori di ritardo molto diversi potrebbero non aver bisogno di una colonna lunga, poiché dovrebbero partizionarsi facilmente. Quando si seleziona la fase stazionaria, è importante sceglierne una che comporti fattori di ritardo diversi per ciascun componente.
La preparazione della colonna è la parte più critica dell'esecuzione di una colonna cromatografica. La massa della fase stazionaria impacchettata nella colonna deve essere almeno 20 volte quella del campione. Se la colonna non ha un disco poroso nella parte inferiore, deve essere imballata con cotone e un sottile strato di sabbia. In questo modo si evita la perdita della fase stazionaria attraverso l'uscita della colonna.
Esistono due metodi per imballare la colonna: il metodo di confezionamento a secco e il metodo del liquame. Nel metodo a secco, la fase stazionaria viene trasferita nella colonna sotto forma di polvere. La colonna viene quindi lavata più volte con la fase mobile/solvente per garantire che il solvente penetri in ogni parte della colonna di gel di silice. Questo metodo, se non eseguito correttamente, può aumentare la probabilità che si formino macchie secche, canali e bolle d'aria nella colonna. Questi difetti influenzeranno la capacità della colonna di partizionare i componenti di una miscela.
Il metodo del liquame fornisce una colonna impaccata più uniforme che non contiene bolle d'aria, aree secche e canali. Per questo metodo, la fase stazionaria viene miscelata con un solvente per formare un impasto omogeneo. Il liquame viene quindi trasferito alla colonna picchiettando lateralmente per eliminare eventuali bolle d'aria formate. Il rubinetto della colonna, se presente, deve essere aperto durante questa fase per consentire il drenaggio del solvente.
<h4>Cromatografia su gel di silice</h4>
Molte proprietà diverse vengono sfruttate per separare una miscela di composti, come le dimensioni, la carica e l'idrofobicità. Una proprietà utilizzata per separare i composti in chimica organica è la polarità. Per questo, il gel di silice e il gel di allumina sono le fasi stazionarie più comunemente usate. Il gel di silice è estremamente polare e forma forti interazioni dipolo-dipolo con composti polari. Inoltre, il gel di silice può formare legami idrogeno con l'analita a causa della presenza di gruppi -OH sulla sua superficie.
I componenti della miscela che sono più polari si legano fortemente al gel di silice e viaggiano lentamente attraverso la colonna, mentre i componenti non polari sono più solubili nella fase mobile e viaggiano rapidamente attraverso la colonna. Pertanto, è essenziale che i componenti della miscela abbiano polarità diverse. I componenti che interagiscono fortemente con la fase stazionaria vengono eluiti facendo scorrere una fase mobile polare attraverso la colonna.
<h4>referenze</h4>
<ol>
	<li style="font-size:17px">Shuler, M.L., Kargi, K., DeLisa, M. (2017). Ingegneria dei Bioprocessi, concetti di base. Upper Saddle River, NJ: Prentice Hall.</li>
	<li style="dimensione del carattere:17px">Harris, D.C. (2015). Analisi chimica quantitativa. New York, NY: W.H. Freeman e compagnia.</li>
</ol>

Cromatografia su colonna

Cromatografia su colonna
Esistono diverse tecniche per purificare e separare i composti in un laboratorio di chimica organica. Uno dei sistemi di separazione più affidabili ...

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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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