Biology Lab-Mr.Willets
Separation of Plant Pigments by Chromatography
A key aspect of photosynthesis is the ability of plant cells to capture the energy from sunlight. This is accomplished by certain pigments including the most common of them- chlorophyll-a. In today's lab, you will separate the 4 pigments found in many plants. The method you will use to separate them is paper chromatography.
Chromatography works by allowing a solvent to rise up a piece of special paper. On the paper are the materials you wish to separate. As the solvent moves up the paper, it dissolves these molecules and carries them up the paper at different speeds (depending on their mass) and thereby separates them. For this to work, the solvent must be able to dissolve the materials you wish to separate. Water would not work on these pigments (water will not dissolve them) so we must use a mixture of ether and acetone.
The plant we will use is spinach which has a high concentration of pigments. Spinach, like most higher plants has 4 pigments: chlorophyll-a, chlorophyll-b, xanthophyll and carotene. Chlorophyll a and b are different shades of green. Xanthophyll is yellow, carotene is yellow/orange (actually it more yellow than orange but work with me on this). With luck, you will be able to see all 4 pigments once separated.
Procedure-
1) Get a piece of chromatography paper from the front desk
2) Get a piece of spinach and a penny. Use the penny to rub some of the pigments from the spinach onto the paper at a spot about 1.5 inches from the bottom as seen on the diagram on the back. This will be demonstrated in class. A dark pigment line is needed for good results. A thinner line is better than a thick one.
3) Bring the paper to the front and get a large test tube from the front desk.
4) Your friendly teacher will add some solvent to your test tube.
5) Place the paper into the test tube. Cover the top of the test tube with a piece of aluminum foil and put the test tube into the test tube rack.
6) Watch as the solvent rises and carries the pigments with it. You should eventually see the 4 pigments being separated. When the 4 pigments have been separated OR the solvent has reached the top of the paper, remove the paper. Pour the solvent back into the flask at the front of the room. DO NOT WASH OUT THE TEST TUBE.
Questions-
1)Use the diagram of the paper strip below to show the location, shape, width and color of each pigment band.
2) In what cell organelle are these pigments found? _______________________
3) What part of a plant would have cells with many of these organelles? _________________
4) Most leaves have yellow and orange pigments. Why don't we normally see them?
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Why are we able to see them in the fall? _____________________________________________
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5) Why wouldn't this experiment work if we used water? ____________________________________
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This is the reason why plain water will not remove what type of laundry stain? _____________________
6) What would have happened to the pigments if the solvent had been high enough to cover the pigment line?
7) The four pigments travelled up the paper at different rates. Why?