Title: Identification and separation of chlorophyll by Thin layer chromatography
Aim/Objective: Identification and separation of plant pigment by TLC
Relevant Information:
Plant pigments are macromolecules produced by the plant, these pigments absorbs specified wavelengths of visible light to provide the energy required for photosynthesis. Chlorophyll is necessary for photosynthesis, light, the wavelengths of light that are not absorbed by the plant pigments and are reflected back to eye. The reflected wavelengths are the colours we see in observing the plants. Plant contains different pigment including: chlorophylls (green), carotenoids (yellow, orange red), anthocyanins (red to blue, depending on pH), betalain (red or yellow).
Thin layer chromatography is useful technique for separating and identifying pigments and other molecules from cell extracts that contain a complex mixture of molecules. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of solvent molecule to the thin layer.
Reagent:
Developing solvent (mobile phase): Take 100ml petroleum ether, 100ml of Iso-propanol and 5 drops of distilled water.
Preparation of TLC chamber:
The solvent should completely cover the bottom of the chamber to the depth of approximately 0.5cm then closed the chamber and shake. It is kept cover so that evaporation does not occur or any change. The composition of developing solvent mixture after 15 min, chamber will be saturated will by the solvent vapors.
Method:
1) Extraction of leaf pigment
1. Using a pistol fresh vegetable leaves or fruit tissue grinded in a mortar containing 22ml of acetone, 3ml of petroleum ether and spatula full of calcium carrbonate (CaCO3)
2. Then filter the extracted pigment using whatman filter paper.
3. Filter is then put into a separating funnel and is mixed with 20ml of petroleum ether and 20 ml of 10% of aqueous NaCl solution and shake well.
4. Separating funnel is kept for few minutes, when the layers are separated the lower layer is allow to drain into a beaker and this phase is thrown away
5. The upper layer is then washed with 5ml of distilled water and again drain off lower layer and discard
6. After the extract is place in erlnmeyer flask and it dried by adding 4 spatula of sodium sulphate
7. Then liquid extract is carefully decanted into round bottle flask and concentrate using a rotating evaporator, to the final volume of about 2-3ml.
2) Application of extract to the TLC plate:
1. With the help of pencil lightly draw an approximately 1.5cm line above at the bottom of TLC plate
2. Take care of the coating of plate should not be scrapped
3. Activate the plate at 100-1200C for 30min in hot air oven.
4. Using a small tip paint brush, apply the concentrated pigment on TLC plate either horizontal line or dot. After each addition dry the plate thoroughly under the stream of cold air and repeat the sample application 2-3 times till very dark marking.
5. The sample spotting line should be kept as a straight as possible for better separation
3) Separation in chromatography chamber
1. Place the TLC plate applied with sample in the TLC chamber containing developing solvent (mobile phase)
2. The level of developing solvent in the TLC chamber should be kept below the sample spot or line.
3. Then close the TLC chamber for separation until solvent front reach the top.
4. Mark the solvent front as soon as plate is removed from the chamber and dry the plate under the stream of hot air using hair dryer.
5. Then mark each individual colour by pencil and measure
the distance from the bottom line. Also measure the distance of solvent fronts from the bottom of line of the plate.
6. Calculate the Rf value of each separated colour and compare the results with standard values.
Observations:
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