Practical 4

Practical 4: Separation of photosynthetic pigments by chromatograph. (Topic 2: Molecular Biology - 2.9 Photosynthesis)

Paper chromatography can be used to separate photosynthetic pigments but thin layer chromatography gives better results.

Introduction

1. The pigments in leaves absorb light energy and convert it to chemical energy. This energy is transferred to and trapped inside chemicals such as glucose and starch.

2. Several different pigments are involved in the absorption of light. This experiment shows how these pigments can be extracted from plants and then separated.

3. Chromatography is a method used to separate chemically similar substances. Once the separation is complete, a chromatogram is formed.

4. The distance that a pigment travels over the thin layer plate depends on two factors:

a) the solubility of that pigment in each of the chemicals in the solvent. The more soluble a pigment the greater the distance it will travel.

b) the adsorption of the pigment to the thin layer plate. The more strongly a pigment is adsorbed to the silica of the plate the more slowly it will travel.

Rf = distance run by pigment (distance from pencil dot to top end of pigment)

distance run by solvent (should be distance between the two pencil lines)

You can identify the principal pigments present by comparing the RF values obtained for each sample to other using data such as the the table of Rf values below. (The RF values vary depending on the solvent used).

Pigment colour Pigment Rf value

orange yellow carotene 0.96

grey a breakdown product 0.70

blue green chlorophyll a 0.58

green chlorophyll b 0.48

deep yellow xanthophyll 0.44

TASK:

Go to the following link.

Virtual lab: http://www.phschool.com/science/biology_place/labbench/lab4/pigsep.html

1) Using the virtual lab write up an aim and protocol for the procedure.

2) Copy the chromatogram and calculate the Rf value for the green pigment present showing clearly your working out.

3) Complete the quiz at the end.