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

La chromatographie sur couche mince, ou CCM, est une technique utilisée pour séparer les composés organiques en fonction des différences de leurs polarités. Un système CCM comporte trois composants : la phase stationnaire, le soluté et le solvant de développement.
La phase stationnaire est la surface sur laquelle les composés seront séparés, qui est généralement composée de gel de silice extrêmement polaire. Les composés ou mélanges analysés sont les solutés. Des solutions diluées de solutés sont repérées sur un bord de la plaque à l’aide de capillaires minces.
Le fond de la plaque est ensuite immergé dans le solvant de développement, qui est généralement un solvant organique moins polaire que la phase stationnaire. Le solvant se déplace vers le haut de la plaque par capillarité. Lorsque le solvant passe à travers les points de soluté, il emporte une partie de chaque soluté avec lui. Ce mélange soluté-solvant est la phase mobile.
Le sommet de la phase mobile est appelé front solvant. Au fur et à mesure que la phase mobile se déplace vers le haut de la plaque, les molécules de soluté et de solvant adhèrent temporairement à la phase stationnaire en fonction de leur polarité.
Les solutés plus polaires sont fortement attirés par la phase stationnaire, de sorte qu’ils collent plus souvent et restent coincés pendant un certain temps. Les solutés moins polaires collent moins souvent et ne restent pas coincés longtemps. Cela signifie que les solutés moins polaires passent plus de temps dans la phase mobile que les solutés plus polaires. En d’autres termes, moins de solutés polaires se déplacent plus rapidement que plus de solutés polaires.
Pour obtenir une bonne séparation entre les composés, le solvant doit être juste assez polaire pour déplacer le soluté le plus polaire. Voici quelques solvants courants par ordre croissant de polarité. Parfois, deux solvants sont combinés pour atteindre la polarité souhaitée.
Parce que la plupart des composés organiques sont blancs ou incolores, les plaques TLC contiennent généralement un composé fluorescent. Sous la lumière UV, les composés organiques apparaîtront sous forme de taches sombres sur la plaque lumineuse.
Une fois les composés visualisés, vous pouvez mesurer la distance qu’ils ont parcourue et calculer le facteur de retard, ou Rf, pour chacun d’eux. Cette valeur est la distance parcourue par le soluté divisée par la distance totale parcourue par la phase mobile.
Si le solvant est choisi correctement, chaque soluté aura un Rf nettement différent. Cela nous permet d’identifier les composés constitutifs des mélanges en faisant correspondre les valeurs Rf aux composés connus.
Dans cet atelier, vous allez utiliser TLC pour déterminer le Rf de plusieurs composés connus, puis vous allez utiliser ces valeurs Rf pour déterminer l’identité d’un composé inconnu.

chromatographie sur couche mince

chromatographie
La chromatographie est une technique utilisée en chimie organique pour séparer les composés d’un mélange en fonction de leurs différences de solubilité entre deux phases différentes. Le concept est similaire à l’extraction liquide-liquide, sauf qu’en chromatographie, les deux phases sont constituées de la phase stationnaire et de la phase mobile. La phase stationnaire est un solide – généralement une bille d’hydrogel à l’échelle microscopique – tandis que la phase mobile est un solvant porteur.
Dans la chromatographie traditionnelle, la phase stationnaire est emballée dans une colonne verticale et le mélange de solution est introduit au sommet de la colonne. Au fur et à mesure que le mélange s’écoule à travers la phase stationnaire, les composés se répartissent entre la phase stationnaire et la phase mobile en fonction de leurs structures et polarités, formant des bandes discrètes. Les solutés qui interagissent faiblement avec la phase stationnaire se déplacent plus rapidement dans la colonne et sortent – ou éluent – en premier. Les solutés qui interagissent plus fortement avec la phase stationnaire se déplacent lentement dans la colonne et éluent plus tard. Les bandes peuvent être collectées individuellement afin d’isoler et de purifier les composés d’un mélange.
Il existe plusieurs types de chromatographie, chacun exploitant une propriété chimique différente pour réaliser la séparation. Par exemple, dans la chromatographie par échange d’ions, les billes de phase stationnaires peuvent être chargées positivement ou négativement, attirant uniquement des molécules de charge opposée. Dans la chromatographie d’exclusion stérique, la phase stationnaire poreuse est composée de billes telles que les polymères d’agarose ou de dextran. Les molécules plus petites peuvent pénétrer plus facilement dans les pores, tandis que les plus grosses molécules s’écoulent au-delà des pores et éluent plus rapidement.
<h4>Chromatographie sur couche mince</h4>
La chromatographie sur couche mince (CCM) est un type de technique de chromatographie qui sépare les composés en fonction de leur polarité. Comme la chromatographie traditionnelle, un système CCM comporte trois composants : la phase stationnaire, la phase mobile et le soluté. Cependant, contrairement à la chromatographie traditionnelle, la phase stationnaire est disposée en une fine couche sur une plaque plutôt que compressée dans une colonne. La TLC utilise le plus souvent du gel de silice polaire, une forme de dioxyde de silicium, comme phase stationnaire. La phase stationnaire forme des liaisons hydrogène en raison des groupes OH à sa surface.
Tout d’abord, une ligne de départ est tracée au bas de la plaque de CCM à l’aide d’un crayon. Les composés ou mélanges analysés sont repérés sur la ligne de départ à l’aide d’un capillaire mince. Ensuite, le fond de la plaque est immergé dans la phase mobile, qui est généralement un solvant organique moins polaire que la phase stationnaire. Le solvant se déplace le long de la plaque par capillarité, passant par les points de soluté et emportant avec lui une partie de chaque composant.
Au fur et à mesure que le solvant remonte sur la plaque, les composants se répartissent entre la phase mobile et la phase stationnaire. Si le composant est polaire, il interagit davantage avec la phase stationnaire polaire. Il se déplace lentement et ne se déplace que sur une courte distance sur la plaque TLC. Si le composant de l’échantillon est moins polaire – et plus soluble dans la phase mobile que dans la phase stationnaire – il interagit davantage avec la phase mobile et se déplace plus loin sur la plaque CCM. L’étendue de la polarité du composant et de la phase mobile sont essentielles pour comprendre et prédire la séparation.
Les plaques TLC contiennent généralement un colorant fluorescent réactif aux UV qui brille sous une source UV de 254 nanomètres. Par conséquent, les plaques CCM peuvent être analysées en les observant sous la lumière UV. Les composés à l’intérieur de la plaque CCM, tels que les solutés d’intérêt, apparaîtront sous forme de taches sombres par rapport à un fond vert. En encerclant les taches avec un crayon graphite, la distance parcourue par les composés par rapport au front du solvant peut être mesurée. La tache du composé organique, si elle n’est pas fluorescente elle-même, masque la fluorescence de la plaque et se manifeste par une tache sombre. Certains composés organiques sont actifs aux UV et émettent de la lumière lorsqu’ils sont exposés à la lumière UV. Il s’agit généralement de composés conjugués, c’est-à-dire avec une alternance de liaisons doubles et simples, et qui peuvent être identifiés par la longueur d’onde émise.
<h4>Facteur de retard</h4>
En analysant le facteur de retard (Rf) d’un composant avec un solvant spécifique, un soluté inconnu peut être déterminé à l’aide de la CCM. Le facteur de retard est le rapport entre la distance parcourue par un composant et la distance parcourue par la phase mobile.
<img src="/CDNSource/lm/labs/62/62_Concepts_1.jpg" font-size>Harris, D.C. (2015). Analyse chimique quantitative. New York, NY : W.H. Freeman et compagnie.

Chromatographie sur couche mince

Chromatographie
La chromatographie est une technique utilisée en chimie organique pour séparer les composés d’un mélange en fonction de leurs différences de solubilité entre ...

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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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