Separation of Plant Pigments by Thin layer chromatography (TLC)

[Asif.Hossain]

Separation Plant Pigments by Thin layer chromatography (TLC)
Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography.Thin layer chromatography is used to separate components of a plant extract

Chemicals and other materials:
1. Petroleum ether
2. Acetone
3. Isopropanol
4. NaCl
5. CaCO3
6. Na2SO4
7. Fresh leaves


Apparatus and glass wares:
1. Rotary evaporator
2. Round bottom flask 100 mL
3. TLC chamber 22 cm × 22 cm × 10 cm
4. TLC silica gel plate  (DC-Alufolien, Kieselgel 60 / Kieselgur F254, Art.5567   Merck)
5. Seperating funnel 100 mL
6  Measuring cylinders 25 mL
7. Measuring cylinder 100 mL
8. Erlenmeyer 100 mL
9. Mortar & pestle & Paint brush


Hazards and safety precautiona:

Petroleum ether is volatile and very flammable. Petroleum ether presents a high fire risk. The toxicity of petroleum ether varies according to its composition. Many of the components are of quite low toxicity, but some formulations may contain chemicals that are suspected carcinogens. Avoid ingestion and inhalation.

Acetone and isopropanol are highly flammable.

Safety glasses and gloves must be worn. The experiment should be performed under a portable fume cupboard giving all-round visibility!


Preparation

Developing solvent (mobile phase):
100 mL of petrol ether, 11 mL of isopropanol and 5 drops of dist. water

Preparation of the TLC chamber:
The developing solvent is placed into a TLC chamber. The solvent should completely cover the bottom of the chamber to a depth of approximately 0.5 cm. The chamber is closed and shaken. It is kept covered so that evaporation doesn't change the composition of the developing solvent mixture. After 15 minutes the chamber will be saturated with the solvent vapor.

Extraction of the leaf pigments:
Using a pestle fresh leaves are grinded in a mortar containing 22 mL of acetone, 3 mL of petrol ether and a spatula tip-ful of CaCO3. The pigment extract is filtered. The filtrate is put into a separating funnel and is mixed with 20 mL of petrol ether und 20 mL of 10% aqueous NaCl solution. The separating funnel is shaken carefully. When the layers have separated the lower layer is allowed to drain into a beaker. This phase is thrown away. The upper layer is washed 3-4 times with 5 mL of dest water. Afterwards the extract is placed in an Erlenmeyer flask and is dried with about 4 spatula tips of Na2SO4. The liquid is carefully decanted into a round bottom flask. Using a rotary evaporator the leaf extract is concentrated to a final volume of about 3 mL.       


Application of the extract to the TLC plate:
With a pencil a line is drawn approximately 1,5 cm from the bottom of the plate. The coating of the plate should not be scraped! Using a paint brush or a Pasteur pipet the leaf extract is applied as a line to the TLC plate. The procedure is repeated until the line is very dark green. The transferred extract is allowed to dry thoroughly after each addition. The line is kept as thin and straight as possible.

Experimental procedure:

The loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom. The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the developing solvent (solvent surface must be below the extract line). The TLC chamber is covered. The TLC plate is allowed to remain undisturbed. When the solvent front has reached three quarters of the length of the plate, the plate is removed from the developing chamber and the position of the solvent front is immediately marked.

Results and discussion:

As the solvent rises by capillary action up through the TLC plate, the components of the pigment mixture are partitioned between the mobile phase (solvent) and the stationary phase (silica gel) due to their different adsorption and solubility strength. The more strongly a given component is adsorbed to the stationary phase, the less easily it is removed by mobile phase. The more weakly a component is adsorbed the faster it will migrate up the TLC plate. On the other hand, the running distance depends on the solubility of the pigment in the solvent. Since the experiment employs a high non-polar solvent (petroleum ether), the pigments that are least polar (carotenes) will be best solved in the non-polar solvent ("similia similibus solvuntus") and will thus have the largest running distance.

 
leaf pigments      color
Carotenes           golden
Pheophytin          olive green
Chlorophyll a        blue green
Chlorophyll b        yellow green
Lutein                  yellow
Violaxanthin         yellow
Neoxanthin          yellow