II. Thin Layer Chromatography
(TLC)
• Describtion
- Identifying substances and testing purity of compounds.
- Quick and needs small amount of sample.
- Stationary phase is a thin layer of Silica gel or Alumina.
- Mobile phase is usually an organic solvent.
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Separation of a mixture using TLC
1. Preparing the chamber: i. Add the mobile phase (1 cm in height) to the developing chamber (Jar).
ii. Place a filter paper in the jar
(contacted to the walls and immersed in the solvent) iii. Close the jar for 30 minutes.
WHY?
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Question
This jar has been standing for 30 minutes. Why is it not ready to be used in a TLC experiment?
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Answer
Although the jar has been standing for 30 minutes, the atmosphere inside it is not saturated with solvent vapor because it was not covered. Any solvent vapor would escape the jar into the room.
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2. Preparing the plates for development: i. Mark the TLC plate with a pencil at 2 cm from its bottom.
ii. Spot the samples on the plate with a capillary glass tube.
iii. If the sample amount is not enough, add more sample.
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Question
What is wrong with the plate shown below?
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Answer
The samples were spotted on the plate too closely together. During the development of the plate, the spots will run together, causing interference.
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3. Developing the plates: i. Hold the plates only from its edges.
ii. Insert the plate in the chamber quickly. WHY?
iii. Sample spots are developed
(separated).
iv. Remove the plate and quickly mark the solvent front with a pencil.
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Question
Predict what will happen when this plate is developed in the chamber.
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Answer
The samples were spotted too low on the plate, and the chamber is too full with the developing solvent. Therefore, when the plate is placed into the chamber, the spots will dissolve in the solvent. The plate cannot be developed.
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4. Identifying the spots: i. If spots can be seen, mark them with a pencil.
ii. If no spots appear, put the plate under an Ultraviolet lamp. When the spots appear, mark them with a pencil.
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Commercial TLC plate after development in normal light
Same TLC plate held under a UV lamp . Note the appearance of additional spots.
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5. Interpreting the data: i. R f values are calculated as in paper chromatography, where:
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Question
Calculate the R f
TLC slide below.
values for the spots on the
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Use the equation:
Answer
Measure the distances from the starting point to the solvent front and from the starting point to the center of each spot, and calculate the R f values:
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16

Question
Which of the samples spotted on the TLC plate below are composed of more than one substance?
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Answer
Samples 1 and 3 are composed of more than one substance, because during the TLC experiment two or more spots separated from the original sample. Sample 2 appear to be composed of one substance.
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