SB0023 Biology : Structure and Function

Preparatory Centre for Science & Technology, UMS

2011/2012

PHOTOSYNTHESIS

Introduction

The chloroplasts in green plants contain many pigments such as chlorophyll a, chlorophyll b,
carotene, and xanthophylls. These pigments have different solubility in certain solvent and they
can be separated by chromatography.

Objectives:

To separate and identify pigments within plant cells by process chromatography

Apparatus:

Blender or cutter
Mortar
Pin
Boiling tube with cork stopper
Chromatography paper strip (Whatman no. 1 or no. 3) with pointed end
Buchner funnel

Material:

Fresh leaves
Acetone 80%
Solvent (mixture of ether petroleum: acetone at 9:1)

Method:

Chloroplast extract preparation
1. Cut 20g of healthy leaves using cutter
2. Grind/Blend the leaves in 50cm
3
acetone 80 %
3. Leave it for 10 minutes
4. Grind again and add more acetone
5. Extract chlorophyll using Buchner funnel into a beaker

Paper chromatography
1. Draw a line across the paper 1 cm up from the triangle. This is you start line.
2. Using the head of small pin as a dropper, place a drop of the chloroplast extract on the
chromatography strip. Let the drop dry. Repeat the process several times (5 times) to
build up a small area of concentrated pigment.
3. Attach the paper strip to the stopper with a pin. Suspend the strip straight into the
boiling tube that contains 10cm
3
solvent. The bottom edge paper should dip into the
solvent, but make sure that the pigment spot is not immersed in the solvent.
4. Let the solvent rise until its front reaches 1cm from the top of the strip.
SB0023 Biology : Structure and Function
Preparatory Centre for Science & Technology, UMS
2011/2012

5. Remove the strip and draw a pencil line to mark the solvent front. Also mark the
pigmented area.
6. Calculated the Rf value for each pigment using the following formula.

Formula:
RF=A/B
A = Distance moved by the pigment from its original spot of the solvent
B= Distance to the solvent front

7. Record your results in the table given below.

Pigment Colour Rf value
Chlorophyll a
Chlorophyll b
Carotene
Xantophyll

8. Explain your result.


Discussion

Why you must use pencil to draw the start line?
What factors are involved in the separation of pigments?
What type chlorophyll does the reaction centre contain? What are the roles of other pigments?
(in chloroplasts).


Conclusion