Biology: Investigation 9.

1: Separation of the Pigments of a Leaf Introduction: From reading the textbook, it can be seen that there are usually more than one pigment in a leaf. These pigments can be separated by using a process called chromatography. This process depends on the fact that different pigments have different molecular weights and have different solubility in certain chemicals. Aim: To separate the pigments in a leaf (using silver-beet as the source). Materials: x1 Strip chromatography paper x1 Boiling tube x1 Cork with a paperclip inserted into it x1 Test-tube stand x1 Bottle chromatography solution (92% petroleum ether and 8% acetone) x1 Mortar and pestle 1mL Sand x1 Bottle acetone x1 Silver-beet leaf x1 Tooth-pick

Method: 1. 2. 3. 4. 5. 6. 7. 8. Cut strip of chromatography paper to fit the boiling tube without touching the sides. This is so the fluid will pass up the paper evenly. Place some chromatography fluid in the bottom of the tube, and cork it so that the atmosphere inside the tube can become saturated with the vapour. Tear up the leaf. Place the pieces in the mortar and pestle with the sand to help break up the tissue and some acetone to extract the pigments, and grind the mixture to a pulp. Filter to remove the excess and sand, or let it settle and then take the extract of pigment off the top of the mixture. Lightly mark a distance from the bottom of the paper about 30mm with a pencil. Place the paper on a raised ledge so that it pokes out in the air. Spot the extract of pigment (using the blunt half of a tooth-pick) on to this overlap this is so that the spot is not spread out, or contaminated with dirt. Keep putting the spot on, letting it dry in between, until the spot of extract is very concentrated. Use the paper-clip to place the paper in the boiling tube so that the bottom of the paper just touches the fluid. Make sure that the pigment spot is just above the fluid line.

9.

Stand the tube upright in the test-tube stand and watch until the chromatography fluid has cleared the pigments.

Results:

Cork Paperclip Test-Tube

Chromatography Solution

Conclusion: Which was the most soluble pigment? According to http://www.chem.purdue.edu/teacher/table_of_contents/UVVUS/UVVI S.Plant%20Pigments_CH.pdf, when doing paper chromatography, compounds that are more soluble move with the chromatography solution, while the less soluble compounds, travel more slowly. Chlorophyll (a and b) is slightly soluble in petroleum/acetone/water and Cartenoids (xanthophylls and carotene) are very soluble in petroleum/acetone/water. This means that the expected result has occurred, in that the

chlorophyll has lagged and the cartenoids have travelled the furthest. However, the solubility of the pigments is not only determined by the solubility of the molecules, but also by the size of the molecules. According to http://www.chemtopic s.com/unit06/pchrom.pdf, the two chlorophylls are identical except that chlorophyll beta has an additional CH=O, therefore chlorophyll b will be the least soluble pigment (as it is a larger molecule and therefore will move more slowly than chlorophyll alpha), followed by chlorophyll a. Again this website states that carotene and xanthophylls are identical except that xanthophylls has an extra OH and HO, making it the larger molecule of the two and therefore will travel more slowly, to be the second most soluble of pigment, with carotene therefore being the most soluble pigment, as it has travelled the furthest, due to the fact that it is the most soluble in petroleum/acetone/water and has the smallest molecule. Which of the pigments would you always find in a leaf? *Plant leaves appear green most of the time as the leaves have a large amount of chlorophyll, which cover up the other colours of the cartenoids (and if present the anthocyanins as well). However, when Autumn rolls round, a decreasing amount of sunlight leads to a decreasing amount chlorophyll production, but its decomposition rate remains the same, meaning that the green pigment in the leaves will disappear to show yellow/orange colour (if dominant amount of cartenoid pigments) or red (if dominant amount of anthocyanin pigments). So it would be fair to say that the cartenoid pigments (carotene and xanthopyll) are always present in the silver-beet leaf, while chlorophyll a and b are not. (According to http://chemistry.about.com/library/weekly/aa082602a.htm) What is the advantage to the plant of having more than one pigment? *It would be advantageous to a plant to have more than one pigment as, for example chlorophyll absorbs red and blue light, reflecting green, but when carotene is also present (carotene blue-green and blue light), together they remove red, blue-green and blue light from sunlight. Carotene functions as an accessory absorber. The energy of the light absorbed by carotene is transferred to chlorophyll, which uses the energy in photosynthesis. Carotene is a much more stable compound than chlorophyll as chlorophyll, in fact is incredibly unstable, as bright sunlight causes it to decompose, meaning that the plant has to continuously break down the chlorophyll and regenerate it. Carotene persists in leaves even when chlorophyll has disappeared. When chlorophyll disappears from a leaf, the remaining carotene causes the leaf to appear yellow. NB: A third pigment, or class of pigments, that occur in leaves are the anthocyanins. Anthocyanins absorb blue, blue-green, and green light. (According to http://www.scifun.org/chemweek/fallcolr/fallcolr.html) Clare Hood 12TE 2010