Thin Layer Chromatography (TLC) is a powerful but very simple, quick and
inexpensive ... method used in organic chemistry for separation and identification
of ...
D E P A R T M E N T O F C H E M I S T RY
CHEM 281L – Organic Chemistry Laboratory 1 Thin Layer Chromatography or TLC (Exp 5) Theory: Thin Layer Chromatography (TLC) is a powerful but very simple, quick and inexpensive analytical method used in organic chemistry for separation and identification of components in a mixture. Because of its simplicity and rapidity, it is often used to monitor the progress of a reaction and to check the purity of products. All chromatography rely on two phases: stationary and mobile. In the case of TLC, the stationary phase is a solid (silica) and the mobile phase is a liquid. This type of chromatography is done on a plate (aluminum or glass), which is coated with a thin layer of the stationary phase (silica). In a TLC run, the sample to be analyzed partitions itself between the two phases. If it has a greater affinity for the mobile phase it will move along the plate faster with the mobile phase and if it has a greater affinity for the stationary phase, it will move slower. Thus, compounds can be separated from each other based on their different polarities. In this experiment, you will use TLC to identify a compound or a mixture of compounds in a given unknown by comparison with the reference compounds. This can be done by simultaneously running the standards with the unknown. The reference compounds are: O OH
A: Naphthalene
B: 2-Naphthol
O C: Naphthoquinone
D E P A R T M E N T O F C H E M I S T RY
Procedure: Obtain 2 TLC plates (one small and one big) from the dispensary. Always hold the plates by the edges and do not touch the chalky side (silica). You will first run the 3 standards on the smaller plate as a practice then you are going to use the larger plate to spot the three reference compounds (A, B and C) as well as your unknown (U) and run the TLC for identification. The TLC experiment will consist of 4 simple stages: 1. Spotting 2. Development 3. Visualization 4. Identification based on Rf values Spotting: Draw a light pencil line gently about 1 cm from the end of each chromatographic plate (see diagram), but do not gouge the surface. Use micro capillaries to spot the smaller plate with the 3 standards, labeling them A, B, C and the other plate with four spots: the 3 standards and your unknown (A, B, C, U). Please use a separate capillary for each compound. Make each spot as small as possible, preferably 0.5 mm in diameter. The less compound you spot the better and cleaner will be the visualization. 0.75~1 cm
1 cm
A
B
C
A
B
C
U
D E P A R T M E N T O F C H E M I S T RY
Development: Prepare a solvent jar with the mobile phase (solvent: petroleum ether/ethyl acetate) by placing enough solvent to cover the bottom to a depth of about 0.5 cm. Place the smaller plate, with the 3 standards spotted, carefully in the solvent jar, making sure that the spots are above the solvent. Cover the jar quickly and allow the solvent to rise up the plate by capillary action (see diagram). When the solvent reaches the top line, take the plate out and allow it to air dry.
D E P A R T M E N T O F C H E M I S T RY
Visualization: Examine the plate under the UV lamp and mark the spots with a pencil. Identification based on Rf values: Develop the bigger plate using fresh solvent, mark the spots and calculate the Rf values (retention factor-sample calculation is shown below) of each spot including that/those of the unknown. Identify the unknown either as a single compound or as a mixture of two compounds by comparing the unknown spot/spots with those of the standards and their Rf values. Complete the report sheet and hand in it at the end of the lab. Rf calculation: The retention factor, or Rf, is defined as the distance traveled by the compound (from spotting line to center of spot) (a) divided by the distance traveled by the solvent (b).
Rf = a/b b Solvent Front
a Spotting Line
