Preparation Prepare the TLC chamber, which can be as simple as a mason jar, with the solvent system. Close the lid to the TLC chamber and allow to equilibrate for about 10 minutes or more. Turn on hot plate to a low setting. Prepare visualization chamber by placing some resublimed iodine crystals into a mason jar and secure the lid.
Procedure Using 2 small pieces of tape, attach the TLC plate to the hotplate such that the bottom of the plate is touching the hotplate. Mark the origin bottom of your plate with a pencil and label each lane. Secure the lid to the chamber and allow the plate to develop. Once the solvent front nears the top of the plate, remove the plate from the TLC chamber, quickly mark the solvent front with a pencil, and allow it to dry. It typically takes minutes for the solvent front to reach near the top of the plate.
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. Thin layer chromatography is done exactly as it says - using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic.
The silica gel or the alumina is the stationary phase. The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later. The mobile phase is a suitable liquid solvent or mixture of solvents.
We'll start with a very simple case - just trying to show that a particular dye is in fact a mixture of simpler dyes. A pencil line is drawn near the bottom of the plate and a small drop of a solution of the dye mixture is placed on it. Any labelling on the plate to show the original position of the drop must also be in pencil. If any of this was done in ink, dyes from the ink would also move as the chromatogram developed.
When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a covered beaker. It is important that the solvent level is below the line with the spot on it. The reason for covering the beaker is to make sure that the atmosphere in the beaker is saturated with solvent vapor. To help this, the beaker is often lined with some filter paper soaked in solvent.
Saturating the atmosphere in the beaker with vapor stops the solvent from evaporating as it rises up the plate. As the solvent slowly travels up the plate, the different components of the dye mixture travel at different rates and the mixture is separated into different coloured spots. The diagram shows the plate after the solvent has moved about half way up it.
The solvent is allowed to rise until it almost reaches the top of the plate. That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase. If all you wanted to know is how many different dyes made up the mixture, you could just stop there.
However, measurements are often taken from the plate in order to help identify the compounds present. These measurements are the distance traveled by the solvent, and the distance traveled by individual spots.
When the solvent front gets close to the top of the plate, the plate is removed from the beaker and the position of the solvent is marked with another line before it has a chance to evaporate.
For example, if the red component traveled 1. If you could repeat this experiment under exactly the same conditions, then the R f values for each dye would always be the same. For example, the R f value for the red dye would always be 0. 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. Colors vary. Potassium permanganate. Works well for all compounds that can be oxidized. Yellow on purple. Yellow or light brown on purple. Bromocresol Green. Yellow spot on blue background. Good general stain, very well with polyhydroxylated and carbonyl compounds.
Blue or green spot. Upon heating, very sensitive! Good general stain, particularly sensitive towards nucleophiles. Varying colors on light pink plate upon heating. Does not work with alkenes, alkynes or aromatic system unless functional groups are present. Phosphomolybdic acid PMA. Very sensitive. Dark green spot on light green plate.
Sensitivity can be enhanced by use of cobalt II chloride. Indoles, amines. Pink or red-violet. Dragendorff-Munier Stain. Amines even the ones that are low in reactivity. Various colors. Due to the fact that all those variables are difficult to keep constant, a reference compound is usually applied to the plate as well. TLC University of Colorado. TLC Macherey Nagel.
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