Use gloves, oil from hands can contaminate the TLC. Mark a line using pencil that is higher than the solvent in the TLC jar and mark down where the sample should be spotted. Use a capillary tube to spot the sample onto the TLC plate. You can spot it several times if the concentration of your sample is not high. Air dry TLC plate after each spotting use a blow drier to evaporate off higher boiling point solvents. Your sample spot should not be wider when mm in diameter. The smaller the diameter of the spot, the better resolution you will observe.
Set the TLC to a slight slant and then close the jar lid. Mark down the location of the solvent front for determining the R f values. Skip to main content. TLC stands for Thin Layer Chromatography It is an analytical technique that can be used to monitor reactions and for the qualitative analysis of complex mixtures and for the identification of unknown compounds.
It is also important for determining the correct solvent system with which to run a column chromatography in. TLC is composed of two phases, a mobile and a solid phase. The solid phase is a thin solid support that usually consists of Alumina or Silica.
The mobile phase is a solvent that moves through capillary action right through the solid phase. In general, the solid phase is usually polar while the mobile solvent is non polar relative to the solid phase. In the first lane of each TLC plate marked "BA" was spotted a dilute sample of the reactant benzyl acetate, while in the third lane of each was spotted the reaction mixture marked "Pr" at different times. In the central lane marked "Co" for the co-spot , both benzyl acetate and the reaction mixture were delivered over top of one another.
Figure 2. A new spot appears below it, representing the benzyl alcohol product. Over time Figure 2. It is apparent from the TLC plates that the reaction was nearing completion at 10 minutes, and was complete at 20 minutes. The TLC demonstrates that the reaction mixture could be worked up after 20 minutes of mixing. To monitor a reaction's progress by TLC, an " aliquot " or tiny sample of the reaction mixture is necessary. If the reaction is run at room temperature or with only mild heating, and the concentration of reactants is conducive to TLC, a capillary spotter can be directly inserted into the flask where the reaction is taking place Figure 2.
A long spotter is ideal if one is available. The aliquot can then be directly spotted on the TLC plate. If the sample spot is not visible before elution it will not be visible afterwards, as compounds diffuse during elution. If the sample is expected to be UV active, and only a faint hint of material is seen on the baseline, the material can be deposited multiple times before elution Figure 2.
If the spots are not allowed to dry in between applications, the spot will be too large. Check the plate under UV light again, and if necessary spot more times. To obtain an aliquot of a refluxing solution, briefly remove the condenser and insert a spotter into the reaction mixture Figure 2.
Immediately re-connect the condenser and adjust the clamps while holding the aliquot. Do not gouge the silica or alumina. Label the areas with pencil where you plan to place the samples Figure 2. A one-inch wide TLC plate can comfortably accommodate three samples have three lanes , and if the spot size is kept small it can fit at maximum five spots. If more than five spots are necessary on one plate, TLC plates can be purchased in sheets and cut wider than one inch.
The lanes should also not be placed too close to one another, or the spots may overlap after elution. A spot is always larger after elution compared to its original size Figure 2. Broadening also occurs as solutes in the mobile phase move at different rates as the flow of eluent is weaker near the adsorbent surface.
This can even sometimes cause compounds to get stuck in adsorbent pores where the flow of eluent is especially weak. Spot broadening means that samples deposited right next to each other on the baseline of a TLC plate will probably bleed together during elution. Spot the TLC plate with sample Obtain a capillary spotter a very thin hollow piece of glass open at both ends. In some institutions, you may need to make your own spotter by stretching a softened pipette.
Place your spotter into the diluted sample you want to analyze to withdraw liquid into the spotter through capillary action. If the liquid level is low in your vial and your spotter short, you may have to tip the vial to withdraw liquid Figure 2.
Keeping the spotter mostly vertical, make a practice "spot" on a paper towel or scrap piece of silica or alumina to familiarize yourself with how the liquid delivers from the spotter. Deliver a very small spot of material on the pencil line of the appropriate lane Figure 2. Don't gouge the silica or alumina with the spotter.
Still keeping the spotter vertical, immediately touch the spotter to a paper towel to expunge all of the liquid remaining in the spotter Figure 2. If the spotter is set down without immediate draining the liquid, air may get into the spotter making it impossible to dispense and use again. Rinse the spotter with acetone or another volatile solvent with which your compounds are soluble, Figure 2.
Place the empty spotter in your rinse vial to withdraw liquid and drain the solvent onto a paper towel. Rinse once or twice before reusing the spotter for other samples. Don't allow the liquid to splash onto the plate. The liquid level must be below the pencil line where the samples are spotted or the compounds will dissolve in the pool of eluent instead of traveling up the plate.
Cap the chamber delicately while keeping it vertical, and don't touch it again until the TLC is complete. Allow the TLC to develop Figure 2. As liquid moves up the TLC plate it will appear transparent and wet.
A dark background will allow the solvent front to be more easily seen. If the eluent is very polar e.
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