Practical 2 - Investigation into the separation of choroplast pigments by chromatography Flashcards
Where are chloroplast pigments located?
On the membranes of the thylakoids and grana
What do chloroplast pigments do in summary?
Harvest light in the light-dependent reactions of photosynthesis, and transfer its envy into the light-independent reactions, in the synthesis of complex organic molecules
What are the 2 major groups of chloroplast pigments in flowering plants?
The chlorophylls
Carotenoids
Types of chlorophyll pigments
Chlorophyll a
Chlorophyll b
Phaeophytin
Which type of pigment is the commonest and is found in all photosynthetic organisms that will be studied?
Chlorophyll a
Where is chlorophyll b found?
In flowering plants
What is phaeophytin?
A type of chlorophyll pigment, which is a break down of chlorophyll a molecule, lacking the central magnesium ion and is seen in flowering plants and in purple sulphur bacteria
What are the types of carotenoids pigments?
a- and B- carotene
Lycopene
Xanthophylls
What colour are both a- and B- carotene?
Orange
What colour is lycopene?
Red
Types of xanthophylls
Lutein and zeaxanthin
What colour do xanthophylls such as lutes and zeaxanthin appear?
Yellow
What is being done in this practical in summary?
Chloroplast pigments will be extracted from the leaves of an angiosperm, separate by chromotography and identified
Flowering plant
Angiosperm
Angiosperm
Flowering plant
What does the distance each pigment travels on the chromatography depend upon?
1.) its solubility in the solvent - more soluble pigments travel further
2.) its absorption by the paper or silica gel - pigments that are absorbed less travel further
What happens to more soluble pigments on a chromatogram?
Travel further
What happens to a pigment that is absorbed more on a chromatogram?
Absorbed less = travel further
Apparatus
Dark green leaves (e.g - stinging nettles, spinach)
Scissors
Sand
Pestle
Mortar
Propanone
Distilled water
2x stopper
Pipette
Vial
Chromatography paper or silica gel chromatography plates
Pencil
Ruler
Capillary tube
Hair drier
2 x boiling tube
Petroleum ether
What are the hazardous substances used in this practical?
Propanone
Petroleum ether
What do we make sure to do with flammable substances?
Keep in the fuming cupboard with the fan on
What do we need to use for the chromatography as we can’t use water?
Chromatography solvent
Method for preparing the pigment solution
1.) chop 1g leaf material finely with scissors and place in the motar
2.) add a pinch of sand and 5cm^3 propanone
3.) grind the leaf fragments to a slurry
4.) place slurry in a boiling tube
5.) add 3cm^3 distilled water, shake vigorously and stand for 8 minutes
6.) add 3cm^3 Petroleum ether mix by gentle shaking and allow layers to separate
7.) collect the upper, petroleum ether layer, which contains the chloroplast pigments in a pipette and transfer to a vial
Steps for preparing the chromatography paper
1.) draw a pencil line across the chromatography paper or plate approximately 2cm from one end
2.) draw chloroplast pigment solution into a capillary tube and put a small spot in the centre of the pencil line. Ensure that the capillary tube does not pierce or tear the chromatography paper
3.) dry and spot as quickly as possible, preventing its spread
4.) repeat steps 2 and 3 \until there is a small but intense spot of pigment
Steps for running the chromatogram
1.) place freshly-made 2:1 propanone:petroleum ether solvent mixture in a boiling tube until it is approximately 5mm deep
2.) without touching the sides of the boiling tube, slide the chromatography paper into the boiling tube so that its end is below the surface of the ethanol but the spot is above, and not touching it
3.) hold the chromatography paper in place with the stopper, folding the paper over the rim of the boiling tube at the top
4.) leave the boiling tube ensuring that it is not moved, until the solvent has climbed up the paper to within 10mm of the top
5.) remove the chromatography paper from the boiling tube and immediately, draw a pencil line across the paper to mark the solvent front
6.0 mark the position of the top of each pigment spot with a pencil
How do we identify the pigments from a chromatogram?
1.) use a ruler to measure the distance from the origin to the solvent front
2.) measure the distance from the origin to the top of each pigment spot
3.) Rf for a h pigment
Distance travelled by pigment
—————————————-
Distance travelled by solvent front
4.) published data allow the pigments to be identified, as Rf is constant for each pigment in a given solvent
Rf
Distance travelled by pigment
———————————————
Distance travelled by solvent front
Where do we measure to when measuring from the origin to the solvent spot and to each pigment spot?
The top every time
Why should chromatogram be kept out of sunlight?
They’re photosensitive, so the colours could fade
