Place the plate into the iodine chamber for visualization and secure the lid. What conclusions can you draw about the polarity of the substances? Discuss one purpose of this technique in research or industry. Place the plate in charged TLC chamber, in front of the wick. Secure lid to chamber and allow the pl…. TLC plate progression. High School. Science Saturday. Learning Resources. Each collection features resources to Know about, Show, Explore, and Relate to an engaging theme for learners and educators.
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Smaller molecules can enter the pores more readily, while the larger molecules flow past the pores and elute faster. Thin-layer chromatography TLC is a type of chromatography technique that separates compounds based on their polarity.
Like traditional chromatography, there are three components of a TLC system: the stationary phase, the mobile phase, and the solute. However, unlike traditional chromatography, the stationary phase is arranged in a thin layer on a plate rather than packed into a column. TLC most often uses polar silica gel, a form of silicon dioxide, as the stationary phase. The stationary phase forms hydrogen bonds due to the OH groups on its surface.
First, a starting line is drawn on the bottom of the TLC plate using a pencil. The compounds or mixture being analyzed are spotted on the starting line using a thin capillary. Then, the bottom of the plate is immersed in the mobile phase, which is usually an organic solvent that is less polar than the stationary phase.
The solvent travels up the plate by capillary action, passing the solute spots and carrying some of each component with it. As the solvent travels up the plate, the components partition to either the mobile phase or the stationary phase.
If the component is polar, it interacts more with the polar stationary phase. It travels slowly and only moves a short distance on the TLC plate. If the component of the sample is less polar — and more soluble in the mobile phase than it is in the stationary phase — it interacts more with the mobile phase and travels farther on the TLC plate.
The extent of the polarity of the component and the mobile phase are essential to understanding and predicting the separation. Compounds within the TLC plate, such as the solutes of interest, will show up as dark spots compared to a green background. By circling the spots with a graphite pencil, the distance the compounds traveled relative to the solvent front can be measured. The spot of the organic compound, if not fluorescent itself, masks the fluorescence of the plate and shows up as a dark spot.
Some organic compounds are UV-active and emit light when exposed to UV light. These are typically conjugated compounds, meaning those with alternating double and single bonds, and can be identified by the wavelength emitted. By analyzing the retardation factor R f of a component with a specific solvent, an unknown solute can be determined using TLC.
The retardation factor is the ratio of the distance traveled by a component to the distance traveled by the mobile phase. The distance traveled by the solute is measured from the starting line to the center point of the spot, and the distance traveled by the mobile phase is measured from the same starting line to the solvent front.
The retardation factor of a compound is dependent on the mobile phase used. The retardation factor is large for compounds that are highly non-polar with a non-polar mobile phase.
However, if anything changes the temperature, the exact composition of the solvent, and so on , that is no longer true. You have to bear this in mind if you want to use this technique to identify a particular dye.
We'll look at how you can use thin layer chromatography for analysis further down the page. You may remember that I mentioned that the stationary phase on a thin layer plate often has a substance added to it which will fluoresce when exposed to UV light.
That means that if you shine UV light on it, it will glow. That glow is masked at the position where the spots are on the final chromatogram - even if those spots are invisible to the eye. That means that if you shine UV light on the plate, it will all glow apart from where the spots are.
The spots show up as darker patches. While the UV is still shining on the plate, you obviously have to mark the positions of the spots by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will disappear again. In some cases, it may be possible to make the spots visible by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures. The chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin.
Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple. In another method, the chromatogram is again allowed to dry and then placed in an enclosed container such as another beaker covered with a watch glass along with a few iodine crystals.
The iodine vapor in the container may either react with the spots on the chromatogram, or simply stick more to the spots than to the rest of the plate. Either way, the substances you are interested in may show up as brownish spots. Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids.
A small drop of the mixture is placed on the base line of the thin layer plate, and similar small spots of the known amino acids are placed alongside it.
The plate is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5. The left-hand diagram shows the plate after the solvent front has almost reached the top.
The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin. There is no need to measure the R f values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colours.
In this example, the mixture contains the amino acids labelled as 1, 4 and 5. And what if the mixture contained amino acids other than the ones we have used for comparison? There would be spots in the mixture which didn't match those from the known amino acids. You would have to re-run the experiment using other amino acids for comparison.
The stationary phase - silica gel. Silica gel is a form of silicon dioxide silica. The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups.
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