Thin Layer Chromatography (TLC)

Video: Introduction to TLC

This video provides an explanation of TLC and demonstrates a general method for the technique. The video below the General Protocol section illustrates how TLC is performed in the labs at UR. Note: If you are off-campus, you must use a VPN connection to the UR network in order to access this video.

General Protocol

Preparing the TLC Sample Solution

1. A TLC sample is prepared by dissolving a compound or sample in a volatile organic solvent. An appropriate concentration should be roughly 5-10% (weight/volume).

Preparing the Developing Chamber

1. Your chamber must be tall enough to be capped while holding the TLC plate you intend to use.

2. Deliver enough of the mobile phase solvent system to form a layer 4-5 mm deep in the TLC chamber.

3. Add a wick (e.g. filter paper) tall enough to reach nearly the top of the chamber.

4. Cap the chamber tightly and allow the solvent to travel all the way up the wick.

5. It is best to allow the chamber to sit for ~15 minutes before using it in order to ensure that the air in the chamber is saturated with the mobile phase solvent.

Preparing the TLC Plate

Note: Handle the TLC plates by the edges and avoid touching the stationary phase with your fingers. 

1. Use a pencil to draw a light starting line (origin) approximately 1 cm from the bottom of the plate. Note that the starting line must be above the solvent level in the TLC chamber. You may use a ruler to help guide you (Figure 1).

Figure 1. Use a ruler to guide you as you lightly draw the origin with a pencil.

2. If you are spotting multiple spots on the same plate, you may label the lanes (locations) in which you intend to spot each sample with a number or other ID code beneath the origin (Figure 2).

Figure 2. Example TLC plate with five samples spotted in individual lanes

3. Samples are applied to the TLC plate using the spotting procedure described below. Note that samples should not be spotted too close to the edge of the plate. Also, you should separate spots of different samples by about 0.5 cm to prevent them from running together during development of the plate.

• Dip the capillary tube (spotter) into the sample solution.

• Quickly and gently touch and remove the end of the micropipette to the surface of the TLC plate on the starting line. Try to make as small a spot as possible, and be careful not to grind the micropipette into the stationary phase.

• If your sample is dilute, you may wait until the spot dries and then reapply the sample to the same spot using the same procedure as many times as necessary. If you do not wait for the spot to dry, reapplication will enlarge the spot and decrease the efficiency of your separation.

• Be sure to clean your spotter between samples by dipping it in a vial containing a small amount of acetone. Allow the spotter to partially fill with acetone, then depress it into a paper towel or Kimwipe to withdraw the acetone. Repeat the washing process at least once more.

• If the samples are visible under UV light, you may observe the plate prior to developing it to ensure that the spots are appropriately aligned and dark enough to see. If the spots are broad or not aligned on the origin, the plate should be discarded and a new one prepared.

Developing the TLC Plate

1. Use forceps to place the TLC plate in the chamber with the starting line toward the bottom (Figure 3). Do not allow the plate to touch the wick because the solvent in the wick will travel onto the stationary phase and ruin the experiment.

Figure 3. Use forceps to insert the TLC plate into the chamber. Ensure the origin is above the mobile phase and do not allow the plate edges to touch the filter paper wick.

2. Close the chamber and allow the solvent to rise up the plate.

3. When the solvent front is approximately 0.5 cm from the top of the plate, remove it from the chamber.

4. Quickly mark the position of the solvent front with a pencil before the solvent evaporates.

Visualizing the Spots

1. Once the solvent has completely evaporated from the plate, you may locate (visualize) the spots. Each spot should be circled with a pencil.

2. There are various methods for visualizing spots because not all compounds can be visualized by a single method. It is always best to use a variety of methods to visualize your TLC plate. A summary of three methods is below:

UV lamp: Hold the dry TLC plate under a UV light and observe any spots that can be seen. Safety Note: Do not look into the UV lamp as serious injury to the eyes can result.

Iodine: Place the dry TLC plate in a chamber than contains iodine crystals for approximately 5 minutes. Be sure to tightly seal the container.

Reagent dip: Use forceps to grip the plate below the starting line. Dip the plate into a jar of reagent so that the entire plate is covered with the solution. Withdraw the plate and allow any excess reagent to drip back into the container. Place the plate on its edge on a paper towel to allow any excess solvent to be withdrawn. Look for any spots to appear. If heating is required to cause visualization, wipe the back of the plate on a paper towel and then place it on a hot plate, in an oven, or expose it to a source of hot air. Watch for spots to appear.

Recording Data

1. Make full‑size sketches of the plates in your notebook labeling any pertinent information (e.g. solvent used). Clearly indicate which spots were detected under the UV lamp and which were detected by other methods.

2. Measure the distance from the starting line to the center of each spot and record on the sketch.

3. Measure the distance from the starting line to the solvent front and record on the sketch.

4. Calculate the Rf values for all spots.

Video: TLC Setup in the UR Labs

This video is a visual introduction to the TLC setup provided in the UR Organic Chemistry Labs.

Video: TLC Theory

This video  provides background on the theory of TLC.