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2.5 Monitoring a Reaction (TLC)

2.5.1 Overview

2.5.2 How to run a TLC plate: Detailed Explanation

Step 1: Prepare the solutions to TLC

Step 2: Gather all the necessary material

Step 3: Prepare the developing container

Step 4: Prepare the TLC plate

Step 5: Spot the plate

Step 6: Check the spots under the UV light

Step 7: Develop the plate

Step 8: Visualize and circle the spots

Step 9: Dip the TLC plate in the visualization reagent

Click here for - Short Explanation

Step 1: Prepare the solutions to TLC

If the sample is not already in solution, dissolve about 1 mg in 1 mL of a volatile solvent such as hexanes, ethyl acetate, or methylene chloride. As a rule of thumb, a concentration of 1% usually works well for TLC analysis. If the sample is too concentrated, it will run as a smear or streak (see troubleshooting section below); if it is not concentrated enough, you will see nothing on the plate. Sometimes you will need to use trial and error to get well-sized, easy to read spots. Here, we retrieved some of the crude product with a pipette to a vial, and then add the solvent DCM (red bottle). You should always identify your vial.

Step 2: Gather all the necessary material

Get everything you need to start the TLC. The jar with its lid, filter paper, microcapillary pipettes (with an acetone vial to clean them), the solvent you will use to develop the plate, TLC plate, the starting material solution (named as SM) and the product solution (named as P), a pencil and a ruler.

Step 3: Prepare the developing container

The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top. Pour solvent into the chamber to a depth of just about 0.5 cm. To aid in the saturation of the TLC chamber with solvent vapors, you can line part of the inside of the beaker with filter paper. Cover the beaker with a watch glass (or with its closure), swirl it gently, and allow it to stand while you prepare your TLC plate.

 

Step 4: Prepare the TLC plate

TLC plates used in the labs are purchased as 5 cm x 20 cm sheets. Each large sheet is cut horizontally into plates which are 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be. Handle the plates carefully so that you do not disturb the coating of adsorbent or get them dirty.

Measure about 0.5 cm from the bottom of the plate. Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin: the line on which you will spot the plate. Take care not to press so hard with the pencil that you disturb the adsorbent. Under the line, mark lightly the name of the samples you will spot on the plate. Leave enough space between the samples so that they do not run together; about 4 samples on a 5 cm wide plate is advised.

Step 5: Spot the plate

Obtain a microcapillary. Clean it with acetone and dry with the paper. Dip the microcap into the solution and then gently touch the end of it onto the proper location on the TLC plate.


You should always hold the microcapillary in a vertical stance. Don't allow the spot to become too large - if necessary, you can touch it to the plate, lift it off and blow on the spot. If you repeat these steps, the wet area on the plate will stay small.

The first 2 spots should be made with the starting material in the SM mark and in the mid mark.

 

DO NOT FORGET to dry the microcapillary, wash it with acetone, and dry it again (and doing all this cleaning procedure twice), when you have to change the product that you will spot!

After washing and cleaning the microcapillary, retrieve from the second solution and spot it in the P mark and on the mid mark.

 

 

Step 6: Check the spots under the UV light

 

You should do a check up on the spots you made, to see if they are over concentrated or if they are really fade, maybe you should add a little more solution to the spot.

 

Step 7: Develop the plate

 

Place the prepared TLC plate in the developing jar, cover it, and leave it undisturbed on your bench top. The solvent will rise up the TLC plate by capillary action. Make sure the solvent does not cover the spot.

Try to leave the TLC plate as most vertical as possible.

 

Allow the plate to develop until the solvent is about half a centimeter below the top of the plate. Remove the jar top and IMMEDIATELY mark the solvent front with a pencil. Allow the plate to dry.

Step 8: Visualize and circle the spots

If there are any colored spots, circle them lightly with a pencil. Hold a UV lamp over the plate and circle any spots you see. Beware! UV light is damaging both to your eyes and to your skin! Make sure you are wearing your goggles and do not look directly into the lamp. Protect your skin by wearing gloves.

If the TLC plate runs samples which are too concentrated, the spots will be streaked and/or run together. If this happens, you will have to start over with a more dilute sample to spot and run on a TLC plate.

Step 9: Dip the TLC plate in the visualization reagent

Afterwards, you should dip slowly the TLC plate in the used visualization reagent.

And then remove the reagent excess and dry it, until you can see the now colored marked spots.
Dispose the solvent in the right container (near the dish washer) and also wash and dry the jar, if you wish to repeat the TLC with other concentration solvent.

Examples of the final TLC plates result.

TLC plate analysis: as we can see, with the 50% solvent the product was able to move further (being the rf valor close to 0.45) than with 25% (rf around 0.15), so the perfect solvent for this product, maybe would be 40% EA/Hex.

 

2.5.3 TLC Solvents choice


2.5.4 Visualization Reagents and Recipes


2.5.5 The Rf value

2.5.6 Troubleshooting TLC

 

Next - 2.6 Quenching a reaction