Narration Japanese Page

16. Thin-Layer Chromatography (TLC)

General Remarks

How to Operate Thin-Layer Chromatography


  1. Take a small amount of a sample with a microspatula and dissolve in a small amount of an appropriate solvent.
  2. Draw the starting line lightly with a pencil at about 10 mm above from a shorter edge of a silica gel plate.
  3. Draw cross marks on the line as chromatography starting points.
  4. Take the sample solution with a capillary, spot the solution lightly on one of the starting points, and dry the spot with a dryer if necessary. Repeat this operation a few times to concentrate the sample in the small spot, which should be less than 2 mm in diameter.
  5. Use a new capillary when a different sample is taken.


  1. Pour the developing solvent into a wide-mouth bottle to about 5 mm in height.
  2. Close the cap and wait a little while until the bottle is saturated with the solvent vapor.
  3. Open the cap and hold the upper end of the TLC plate with tweezers. Place the TLC plate in the bottle so that the bottom of the plate is dipped in the solvent, and the top of the plate is leaned against the wall of the bottle. The solvent should be drawn straight upright.
  4. Finish the development when the solvent front reaches at about 10 mm below the upper end of the TLC plate.

Calculation of the Rf value

  1. Take out the TLC plate and immediately mark the front line of the developing solvent with a pencil.
  2. Wait for the solvent to evaporate or dry the TLC plate with a dryer.
  3. Turn on a UV lamp. Trace the outlines of the spots detected with UV ray with a pencil.
  4. Record the shape and color of the spots.
  5. Calculate Rf value.
    Rf = a/b
    a = The distance from the starting point to the gravity center of the sample spot.
    b = The distance from the starting point to the front of the developing solvent.


The developing solvent is drawn into silica gel when a TLC plate end is dipped in the solvent. Sample compounds put on the starting point move on the TLC plate along with the developing solvent, during which the compounds are repeatedly adsorbed and desorbed by the silica gel. Less polar compound is adsorbed weakly by the silica gel and thus moves fast. On the other hand, more polar compound is adsorbed tightly by the silica gel and thus moves slowly. As a result, the former travels farther than the latter from the starting point. A mixture of compounds can be separated based on the difference of their polarity. TLC is utilized for checking the purity of a compound and the progress of a reaction.