Thin layer chromatography is a rather simple procedure and it is because of this simplicity that it is such a valuable and common analytical tool in the organic chemistry lab.
- Obtain a plate.
Obtain a TLC plate from the lidded jar of plates. Be sure to handle the plate with forceps, as overhandling the plate with your hands will degrade the silica. It is also important to remember that these slides are silica coated GLASS which means that you can cut yourself. They do not have razor sharp edges, but be careful when handling them. If you happen to break one, be sure to clean it up appropriately. All used TLC plates go in the glass disposal container.
- Mark the plate.
Once you have a plate, the next step is to label and mark it. With a fine tip pencil (do not use a pen, as the dye may separate and run along with your sample), draw a horizontal line across the width of the TLC plate, about 1 cm from one end. This is the starting point of the TLC run where you will do your sample spotting. Label underneath this line the locations of your sample spots. Whatever method works for you to remember what you spotted is fine. Keep in mind that your markings should be very distinct, as the solvent may smear your penciling. - Spot the plate.
With a capillary tube, obtain a small amount of sample. Your sample must either be a liquid or in solution. If your sample is a solid and you must dissolve it in solution, it really doesn't matter what solvent you use to do this. It does not have to be the same solvent that you use to develop the slide.Place one end of the tube into the sample. Through capillary action the sample will be drawn into the tube. Place your finger over the other open end of the tube to prevent the sample from falling out. Hold the tube slightly above the location on the starting line that you had previously marked for spotting. Carefully allow air into the opposite end of the capillary tube by slightly raising your finger that is covering the opening. Allow only a single drop to fall on the plate -- the smaller the better. If the sample is not leaving the tube, slight tapping on the opposite end should coax out a droplet. Allow the spot to evaporate. Repeat this procedure 2-3 times. Concentration of your spotting will definitely affect your TLC run and it is a matter of experimentation to determine what will give you the best results. Repeat this procedure for the remaining samples. The plates only allow for 2-3 samples to be spotted to provide for adequate spacing between sample spots. Over spotting or inadequte spacing between samples can lead to their bleeding into each other during the run.
- Put solvent in the development jar.
Pour approximately 5 mL of solvent in the bottom of a glass development jar. Choosing a solvent is covered in another portion of this website. Replace the lid on the development jar until you are ready to perform the run. This prevents evaporation of your solvent from the jar. INSERT SOMETHING HERE ABOUT THE FILTER PAPER....
- Begin the run.
Using forceps, carefully transfer the TLC plate into the development jar. Place the marked end down into the solvent, and be careful not to splash the solvent onto the plate. We want the solvent to rise up the plate evenly. Replace the lid on the development jar. The solvent will rise up the plate via capillary action and should begin to take the sample with it.
- Monitor the run.
The time that it takes for the solvent to rise up the plate is dependent upon your choice of solvent. Generally a run will take only a few minutes, but you must monitor the solvent front to be sure that it does not reach the end of the plate. This could result in your sample being carried to the end of the plate and an unusable TLC run. A good guideline to follow is to allow the solvent front to reach a point about 1 cm from the end of the slide.
- Remove the plate.
Once the solvent front has come to a point about 1 cm from the plate's edge, unscrew the lid from the development jar and remove the plate with a pair of forceps. Place the still wet slide on a paper towel underneath a hood so that the solvent may evaporate off. However, before this occurs, be sure to use a pencil to mark the extent of the solvent front on the plate. You will know when the slide is adequately dry because it will no longer have the darker, wet quality that it had when removed from the development jar. Properly dispose of the remaining solvent.