Chromatography is the practice of separating out different species in a solution by exploiting their differing desires to to remain in the mobile or stationary phases. If a species has a greater affinity for the stationary phase, it won’t advance far up the slide whereas species which prefer to remain in the mobile phase will move further up.
In TLC, a small amount of the substance to be examined is placed about 2 cm from the bottom of the silica slide. In addition, if the substance is believed to contain one or more particular chemicals, pure samples of these may be placed at the same level on the slide. The slide is placed in a beaker containing a small quantity of solvent and a filter paper partially immersed in the solvent to produce a saturated atmosphere. It is important the solvent in the beaker does not reach the samples initially. Rather the solvent advances up the silica slide by capillary action.
After a period of time, the slide is removed from the sealed beaker and examined. It may be necessary to examine the slide under UV light to determine where the different species have ended up. Multiple spots on the same vertical line will indicate the original substance contained multiple chemicals. Comparison to the reference spots will help identify the spots. The distance travelled by each species and by the solvent is recorded and the retention factor rf, defined as the ratio of the distance traveled by the compound(s) to the distance traveled by the solvent, is calculated.
It is important to specify the solvent used and, in some cases, it may be necessary to try several different solvents.