Separating and Analysis of Photosynthetic Pigments in
a leaf using Chromatography.
Apparatus
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freshly picked leaves of Tradescantia, if possible, or spinach (a last resort, grass).
propanol (20ml per student) [Safety Data; http://ptcl.chem.ox.ac.uk/~hmc/hsci/chemicals/2propanol.html]
Special Solvent (90% petroleum ether + 10% propanone), (5ml per student)
Dropping pipette
Capillary tube
pestle and mortar
boiling tube with bung
small quantity of washed and dried sand
Kettle – to make a beaker of boiling water.
access to an iced water bath, with lid.
access to colorimeter
access to centrifuge
Aim
To see that the green colour in plants is not due to a single pigment but three or more.
To indentify the following pigments; Carotenoids (hydrophobic), Xanthophyll
(hydrophilic), Chlorophyll A (hydrophobic) Chlorophyll B (hydrophobic)
Method A - Extract the Chlorophyll
Wear safety glasses, No flames must be used during this lab.
1. Kill the cells of your leaf by holding it in boiling water for about 20 seconds.
2. Finely cut about 10g of leaf material, excluding the midrib and stem using
scissors.
3. Crush the leaves with 20ml propanone and a pinch of sand with a pestle and
mortar.
4. Decant the solution using a glass rod to hold the leave debris.
5. Centrifuge the green solution to clear all the remaining suspended matter.
6. Pour the supernatant into a small glass tube, but a bung in the tube and store in ice
in the dark while you read the rest of the sheet.
Method B - Separating and identifying the pigments using Chromatography
Only ever touch the edges of a piece of chromatography paper.
1. Remove the strip of chromatography paper from the tube and draw a pencil line
across, 2.5cm from the end.
2. Put the special solvent (propanone 90% and petroleum ether 10%) into the tube
up to 1.5cm depth.
3. Replace the bung and allow the air in the tube to become saturated with solvent
for 10 mins.
4. Put the strip of paper onto two glass rods so that the "loading point" on your line
is not touching the bench.
5. Put a tiny spot of chlorophyll mixture onto the line on the paper, and allow each
to dry. Add 10 more drops SLOWLY for best results.
6. Put the paper back into the tube, avoiding letting it touch the edges of the tube,
replace the bung
7. Put the tube in a dark iced water bath and wait for the solvent to almost reach the
bung. (keep looking)
8. Remove the paper and mark the solvent front with a PENCIL line.
9. Hang up to dry in a warm dark place.
10. When dry, mark the individual pigment spots on the paper, and store it in a dark
place for later analysis. This will be assessed for data analysis. You can compare
your calculations with the text book walues.
Data Analysis and Presentation
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Present the results of both experiments in an appropriate way which shows your
findings clearly.
Don't forget error analysis.
Do a calculation / processing to help identify the pigments.
Conclusion and Evaluation
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Describe the pigments you have found and their colours. Include reference to
your results.
Compare what you find to any text book which give details of the photosynthetic
pigments found in leaves.
Explain the degree of accuracy of your experiment, and any limitations.
Suggest a way to improve anything you have noticed that is not accurate.
Rf values link
Photosynthetic pigments – Using rF values to identify them.
Here is a paper choromatogram from an experiment to separate the photosynthetic
pigments from spinach.
Rf Value table for the solvent used (90% propanol, 10% ether)
Pigment
Rf value
Carotene
0.94
Xanthophyll
0.89
Chlorophyll a
0.46
Chlorophyll b
0.22
Results
1. What is an Rf value? How do you calculate it?
2. Calculate the Rf value for each of the bands in the above chromatograph.
3. Using the information in the table above work out which band is which pigment.
4. What colour is each of the pigments?
Questions
1. Describe what happened to the original spot of plant pigments?
2. Which of the 4 pigments migrated the furthest and why?
3. Which type of chlorophyll was the most soluble?
4. Explain why leaves change colour in autumn?
5. What is the function of plant pigments in photosynthesis?
Evaluation
Complete the table below
Error in the experiment
How did this effect the
results
How to eliminate the error
in a further experiment
A little theory (and revision)
Photosynthetic pigments are present in the chloroplasts of plant cells and absorb light.
There are a number of pigments which absorb different wavelengths of light. Green light
is mostly reflected. The light absorbed is used to split water into hydrogen and oxygen
and then to create carbohydrate from carbon dioxide and hydrogen. This process is called
photosynthesis. The pigments are attached to membranes in the chloroplast called
thylakoids and grana.
Some pigments are hydrophilic and others hydrophobic. Hydrophilic molecules are polar
and have electrical charges (e.g. phosphate of phospholipid). They are soluble in water
and ethanol and insoluble in lipid, oils, ether. Hydrophobic molecules are non-polar and
have no electrically charged areas. (e.g. fatty acid chains in phospholipids). They are
insoluble in water and propanol. Soluble in oils and lipids.
We can separate them using two solvents, one hydrophilic and the other hydrophobic.
For these we will use "Petroleum ether" and "Propanone". The hydrophilic pigments will
dissolve in the hydrophilic solvent and vice versa.
If this experiement is not used for assessment this sheet can be used for
results