5.6.4 Practical: Investigating Photosynthetic Pigments with Chromatography Flashcards
What can chromatography be used for with photosynthetic pigments?
Chromatography can be used to separate and identify chloroplast pigments that have been extracted from a leaf
What is chromatography?
Chromatography is an experimental technique that is used to separate mixtures:
Different components within the mixture travel through the material at different speeds due to their size and charge
This causes the different components to separate
A retardation factor (Rf) can be calculated for each component of the mixture
What is paper chromatography?
Paper chromatography – the mixture of pigments is passed through paper (cellulose)
What is thin-layer chromatography?
Thin-layer chromatography – the mixture of pigments is passed through a thin layer of adsorbent (eg. silica gel), through which the mixture travels faster and separates more distinctly.
Describe a method to investigate photosynthetic pigments with chromatography.
Draw a straight line in pencil approximately 1cm above the bottom of the filter paper being used
Do not use a pen as the ink will separate into pigments within the experiment and obscure the results
Cut a section of leaf and place it in a mortar
It is important to choose a healthy leaf that has been in direct sunlight so you can be sure it contains many active photosystems
Add 20 drops of acetone and use the pestle to grind up the leaf sample and release the pigments
Acetone is an organic solvent and therefore fats, such as the lipid membrane, dissolve in it
Acetone and mechanical pressure are used to break down the cell, chloroplast and thylakoid membranes to release the pigments
Extract some of the pigment using a capillary tube and spot it onto the centre of the pencil line you have drawn
Suspend the paper in the chromatography solvent so that the level of the solvent is below the pencil line and leave the paper until the solvent has reached the top of the paper
The mixture is dissolved in the solvent (called the mobile phase) and the dissolved mixture then passes through a static material (called the stationary phase)
Remove the paper from the solvent and draw a pencil line marking where the solvent moved up to
The pigment should have separated out and there should be different spots on the paper at different heights above the pencil line, these are the separate pigments
Calculate the Rf value for each spot
How do you calculate the Rf value and where do you measure to?
Rf value = distance travelled by component (pigment) ÷ distance travelled by the solvent
Always measure to the centre of each spot
What are the expected results?
Carotenoids have the highest Rf values (usually close to 1)
Chlorophyll b has a much lower Rf value
Chlorophyll a has an Rf value somewhere between those of carotenoids and chlorophyll b
Small Rf values indicate the pigment is less soluble and/or larger in size
What does the Rf value demonstrate?
The Rf value demonstrates how far a dissolved pigment travels through the stationary phase
Molecules with a higher affinity to the stationary phase, such as large molecules, will travel slower and therefore have a smaller Rf value
Molecules that are more soluble in the mobile phase will travel faster and therefore have a larger Rf value
What are the limitations of this experiment?
Paper chromatography is not as specific as other chromatography techniques
It is sufficient to separate and distinguish different pigments and to calculate their Rf value
Chromatography does not give data on the amount of each pigment present or the wavelengths that they absorb
Colorimetry can be used to calculate these values
