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

La cromatografia su strato sottile, o TLC, è una tecnica utilizzata per separare i composti organici in base alle differenze nelle loro polarità. Ci sono tre componenti di un sistema TLC: la fase stazionaria, il soluto e il solvente di sviluppo.
La fase stazionaria è la superficie su cui verranno separati i composti, che di solito è composta da gel di silice estremamente polare. I composti o miscele analizzati sono i soluti. Le soluzioni diluite dei soluti vengono individuate su un bordo della piastra utilizzando sottili capillari.
Il fondo della piastra viene quindi immerso nel solvente di sviluppo, che di solito è un solvente organico meno polare della fase stazionaria. Il solvente risale la piastra per azione capillare. Quando il solvente passa attraverso le macchie del soluto, porta con sé una parte di ciascun soluto. Questa miscela soluto-solvente è la fase mobile.
La parte superiore della fase mobile è chiamata fronte del solvente. Mentre la fase mobile viaggia lungo la piastra, le molecole di soluto e solvente si attaccano temporaneamente alla fase stazionaria in base alla loro polarità.
I soluti più polari sono fortemente attratti dalla fase stazionaria, quindi si attaccano più spesso e rimangono bloccati per un po'. Meno soluti polari si attaccano meno spesso e non rimangono bloccati a lungo. Ciò significa che un minor numero di soluti polari trascorre più tempo nella fase mobile rispetto a un maggior numero di soluti polari. In altre parole, meno soluti polari viaggiano più velocemente di più soluti polari.
Per ottenere una buona separazione tra i composti, il solvente dovrebbe essere abbastanza polare da spostare il soluto più polare. Ecco alcuni solventi comuni in ordine crescente di polarità. A volte, due solventi vengono combinati per ottenere la polarità desiderata.
Poiché la maggior parte dei composti organici sono bianchi o incolori, le lastre TLC di solito contengono un composto fluorescente. Sotto la luce UV, i composti organici appariranno come macchie scure sulla piastra luminosa.
Una volta visualizzati i composti, è possibile misurare la distanza percorsa e calcolare il fattore di ritardo, o Rf, per ciascuno di essi. Questo valore è la distanza percorsa dal soluto divisa per la distanza totale percorsa dalla fase mobile.
Se il solvente viene scelto correttamente, ogni soluto avrà un Rf nettamente diverso. Questo ci permette di identificare i composti componenti nelle miscele abbinando i valori di Rf ai composti noti.
In questo laboratorio, utilizzerai TLC per determinare l'Rf di diversi composti noti, quindi utilizzerai quei valori Rf per determinare l'identità di un composto sconosciuto.

cromatografia su strato sottile

cromatografia
La cromatografia è una tecnica utilizzata in chimica organica per separare i composti in una miscela in base alle loro differenze di solubilità tra due diverse fasi. Il concetto è simile all'estrazione liquido-liquido, tranne che in cromatografia, le due fasi sono costituite dalla fase stazionaria e dalla fase mobile. La fase stazionaria è un solido, tipicamente una perla di idrogel su microscala, mentre la fase mobile è un solvente vettore.
Nella cromatografia tradizionale, la fase stazionaria viene impacchettata in una colonna verticale e la miscela in soluzione viene introdotta nella parte superiore della colonna. Mentre la miscela scorre attraverso la fase stazionaria, i composti si dividono tra la fase stazionaria e la fase mobile in base alle loro strutture e polarità, formando bande discrete. I soluti che interagiscono debolmente con la fase stazionaria si muovono più rapidamente attraverso la colonna ed escono per primi, o eluiscono. I soluti che interagiscono più fortemente con la fase stazionaria si muovono lentamente attraverso la colonna ed eluiscono successivamente. Le bande possono essere raccolte singolarmente per isolare e purificare i composti in una miscela.
Esistono diversi tipi di cromatografia, ognuno dei quali sfrutta una diversa proprietà chimica per ottenere la separazione. Ad esempio, nella cromatografia a scambio ionico, le perle di fase stazionaria possono essere caricate positivamente o negativamente, attirando solo molecole con carica opposta. Nella cromatografia ad esclusione dimensionale, la fase stazionaria porosa è composta da perle come polimeri di agarosio o destrano. Le molecole più piccole possono entrare più facilmente nei pori, mentre le molecole più grandi fluiscono oltre i pori ed eluiscono più velocemente.
<h4>cromatografia su strato sottile</h4>
La cromatografia su strato sottile (TLC) è un tipo di tecnica cromatografica che separa i composti in base alla loro polarità. Come la cromatografia tradizionale, ci sono tre componenti di un sistema TLC: la fase stazionaria, la fase mobile e il soluto. Tuttavia, a differenza della cromatografia tradizionale, la fase stazionaria è disposta in uno strato sottile su una piastra piuttosto che impacchettata in una colonna. TLC utilizza più spesso il gel di silice polare, una forma di biossido di silicio, come fase stazionaria. La fase stazionaria forma legami idrogeno a causa dei gruppi OH sulla sua superficie.
Per prima cosa, una linea di partenza viene tracciata sul fondo della lastra TLC usando una matita. I composti o le miscele analizzati vengono individuati sulla linea di partenza utilizzando un sottile capillare. Quindi, il fondo della piastra viene immerso nella fase mobile, che di solito è un solvente organico meno polare della fase stazionaria. Il solvente risale la piastra per azione capillare, passando attraverso le macchie di soluto e portando con sé parte di ciascun componente.
Mentre il solvente risale la piastra, i componenti si dividono in fase mobile o stazionaria. Se il componente è polare, interagisce maggiormente con la fase stazionaria polare. Viaggia lentamente e si muove solo per un breve tratto sulla piastra TLC. Se il componente del campione è meno polare – e più solubile nella fase mobile rispetto alla fase stazionaria – interagisce maggiormente con la fase mobile e viaggia più lontano sulla piastra TLC. L'entità della polarità del componente e la fase mobile sono essenziali per comprendere e prevedere la separazione.
Le lastre TLC contengono in genere un colorante fluorescente reattivo ai raggi UV che si illumina sotto una sorgente UV di 254 nanometri. Pertanto, le lastre TLC possono essere analizzate osservandole sotto la luce UV. I composti all'interno della piastra TLC, come i soluti di interesse, appariranno come macchie scure rispetto a uno sfondo verde. Circondando le macchie con una matita di grafite, è possibile misurare la distanza percorsa dai composti rispetto al fronte del solvente. La macchia del composto organico, se non è essa stessa fluorescente, maschera la fluorescenza della lastra e si presenta come una macchia scura. Alcuni composti organici sono attivi nei raggi UV ed emettono luce se esposti ai raggi UV. Si tratta di composti tipicamente coniugati, cioè quelli con legami doppi e singoli alternati, e possono essere identificati dalla lunghezza d'onda emessa.
<h4>Fattore di ritardo</h4>
Analizzando il fattore di ritardo (Rf) di un componente con un solvente specifico, è possibile determinare un soluto sconosciuto utilizzando la TLC. Il fattore di ritardo è il rapporto tra la distanza percorsa da un componente e la distanza percorsa dalla fase mobile.
<img src="/CDNSource/lm/labs/62/62_Concepts_1.jpg">
La distanza percorsa dal soluto viene misurata dalla linea di partenza al punto centrale del punto e la distanza percorsa dalla fase mobile viene misurata dalla stessa linea di partenza al fronte del solvente. Il fattore di ritardo di un composto dipende dalla fase mobile utilizzata. Il fattore di ritardo è elevato per i composti che sono altamente apolari con una fase mobile non polare. Valori bassi del fattore di ritardo si osservano per le componenti polari con una fase mobile non polare.
Nel caso di una fase mobile altamente non polare, alcune componenti polari potrebbero non muoversi affatto. Ciò si traduce in un fattore di ritardo estremamente basso e in una separazione insufficiente. Una fase mobile altamente polare fa sì che il composto si muova con il solvente e produce un fattore di ritardo estremamente elevato. Ciò si traduce in una separazione molto ridotta tra i componenti.
Affinché la separazione sia efficace, i fattori di ritardo dei componenti dovrebbero essere distanti circa 0,3 - 0,7. Per trovare la fase mobile efficiente, vengono impiegati tentativi ed errori. Spesso, una miscela di due solventi si rivela più efficace.
<h4>referenze</h4>
<ol>
	<li style="dimensione del carattere:17px">Harris, D.C. (2015). Analisi chimica quantitativa. New York, NY: W.H. Freeman e compagnia.</li>
</ol>

Cromatografia
La cromatografia è una tecnica utilizzata in chimica organica per separare i composti in una miscela in base alle loro differenze di solubilità tra ...

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Thin-Layer Chromatography

tlc separation by polarity

Thin-layer chromatography (TLC) separates compounds in a mixture by polarity. It uses the same basic parts as other chromatography methods: a stationary phase, a mobile phase, and the solute. In TLC, the stationary phase is a thin coating on a plate instead of material packed into a column.

The stationary phase in most TLC systems is polar silica gel, a form of silicon dioxide. Its surface has OH groups that can form hydrogen bonds. The sample is first spotted on a pencil start line with a thin capillary. Then the bottom of the plate is placed in the mobile phase, which is usually an organic solvent that is less polar than the stationary phase.

As the solvent moves up the plate by capillary action, it carries the sample components with it. Each compound then partitions between the mobile phase and the stationary phase. A polar component interacts more with the polar stationary phase, so it moves more slowly and stays near the start. A less polar component is more soluble in the mobile phase, so it travels farther up the plate.

TLC plates often include a UV-reactive fluorescent dye that glows under 254 nm UV light. The compounds appear as dark spots against the green background, and the spots can be circled with a graphite pencil so their movement can be measured. Some organic compounds are UV-active and emit light when exposed to UV light. These are often conjugated compounds, which have alternating double and single bonds, and they can be identified by the wavelength they emit.

The retardation factor, or Rf, helps identify an unknown solute from TLC results. It is the ratio of the distance traveled by the compound to the distance traveled by the solvent front. The compound distance is measured from the start line to the center of the spot, and the solvent distance is measured from the same start line to the solvent front. Rf depends on the mobile phase used.

Rf values are high for highly non-polar compounds with a non-polar mobile phase, and low for polar compounds with a non-polar mobile phase. If the mobile phase is too non-polar, some polar compounds may not move at all. If it is too polar, the compound may move with the solvent and the separation may be poor. For an effective separation, the Rf values of the components should be about 0.3 to 0.7 apart. Choosing the best mobile phase usually takes trial and error, and a mixture of two solvents often works well.

TLC Separation by Polarity

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TLC Separation by Polarity

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