EXPERIMENT 8 Objective. To separate and study the plant pigments by paper chromatography.

 

                               EXPERIMENT 8

Objective. To separate and study the plant pigments by paper chromatography.

REQUIREMENTS

Fresh spinach leaves, chromatographic paper (Whatman No. 1), a wide-long test tube, a split cork, mortar and pestle, petroleum ether, acetone, funnel, beaker, filter paper, capillary tube sand etc.

PROCEDURE

1.Grind a few spinach leaves with little find sand and about 5 ml of acetone in a mortar and pestle. Filter it to get acetone extract of the leaf pigments.

2. Take a narrow strip of chromatographic paper (Whatman No. 1). Cut one end of the strip into a narrow notch.

3. Put a drop of the pigment extract in the middle of the strip near the notch with the help of capillary tube.Allow the drop to dry and repeat till four or five drops are placed on the paper.

4. Take the test tube and pour about 5 ml of ether-acetone solvent (9 ether: 1 acetone) in it. Now hang the pigment extract loaded chromatographic strip in the test tube with the help of a split cork, in such a way that the loading spot lies about 1 cm above the solvent level.

5. Make the cork air tight and place the test tube undisturbed for some time; when solvent rises about 3/4th of the strip, take out the strip carefully and let it dry.

OBSERVATIONS

The dried chromatographic paper strip shows four distinct pigment bands. Different leaf pigments can be identified by their colours. Measure the distance of each pigment band from the loading spot and also the distance travelled by the solvent.

CALCULATIONS

R, value of each pigment spot can be calculated as follows:

=

RESULT

The uppermost orange yellow band corresponds to carotene. The yellowish band below it marks the xanthophylls.The third from above dark green band represent chlorophyll a. The lower most yellowish green band is that of chlorophyll b.

PRECAUTIONS

1.Spinach leaves should be fresh and green.

2.The loading spot should be small and concentrated.

3.The loading spot should be placed 2-3 cm away from the tip of the notch.

4. While hanging the strip in the test tube, the loading strip should remain about 1 cm above the solvent level.

5. The chromatographic paper strip should not touch the walls of the test tube.

6. The chromatographic paper strip should not be dried in sunlight.