Plant Pigment Chromatography:- M3 ( LAB REPORT )

Plant Pigment Chromatography

Aim: To separate pigments from leaves of a green plant using paper chromatography and to determine the wavelength at which energy is absorbed by the individual pigments using spectrophotometry.

Introduction: All cells must constantly consume fuel molecules to maintain themselves, grow, and reproduce. Fuel molecules such as glucose constitute an immediate source of energy for biological work that can be released by catabolic cell processes. However it is necessary that life on earth have a constant source of energy that can be harvested and used to generate complex fuel molecules from simple starting materials. The ultimate energy source upon which all life forms depend is visible light from the sun. There are four different pigment groups present in leaves of photosynthesizing plants. Studies indicate that only the chlorophyll IS involved in the actual absorption of light energy and later conversion to chemical energy of living cells. The other pigments also absorb light energy, but it is transferred to the chlorophyll for conversion to chemical energy.

Requirements: 2 or 3 fresh spinach leaves, wooden ruler, 600 mL beaker, plastic wrap, chromatography paper or filter paper, pencil, coin, 50 mL graduated cylinder, test tubes, scissors, stapler, goggles, cork stopper.

Procedure:

1.  Obtain a 50 ml graduated cylinder with 5ml of solvent in the bottom
   Cut a piece of filter paper or chromatography paper and the edges must be
   Straight that it should reach to the solvent
2. With a pencil lightly make a line 2 cm from the bottom edge of the paper.
3. Select large dark green spinach leaves.
4. Place a leaf over the pencil line leaving on each end to align the ruler.
5. Place the widest side of a wooden ruler (without metal edge) over the leaf so that it covers the pencil line on either end.
6. Using a coin, press down firmly and roll along the ruler edge several times to form a definite green line.
7. Allow the green line to dry
8. Move leaf down and repeat several times until the pencil line is covered completely with a narrow green band. Be careful not to smear this green line.  
9. Staple top and bottom to form a cylinder.
10. Place paper cylinder in beaker with the green band down.
11. The solvent should not touch the green line.
12. Cover the beaker tightly with a piece of plastic wrap being careful not to slosh solvent.
13. Allow to stand undisturbed for 5 minutes.
14. After five minutes lift up corner of wrap and reseal. This will reduce the vapour pressure inside the beaker.
15. Observe the solvent movement and band separation.
16. When the solvent front is within 1 cm of the upper edge of the paper, remove the cylinder from the beaker. Mark the edge of the solvent front with a pencil.
17. Measure distance from the first pencil line to the solvent front. Then measure the distance from the pencil line to the highest point of each colour band and the original pencil line band. Record your results.

Observation:

DATA TABLE
Band No.	Distance Moved (mm)	Band Colour
1	12 mm	olive green
2	21 mm	bright green
3	27 mm	bright yellow
4	53 mm	yellow-orange
Solvent front moved 53 mm