Paper Chromatography To Separate Fall Leaf Colors: A Science Fair Project

Introduction
In the fall (Autumn) season leaves change their green color to red, yellow or orange. Where do these colors appear from? Can they be separated and identified? These colors are due to substances called leaf pigments. The leaf pigments can be separated by paper chromatography. Plants use sunlight and convert water and carbondioxide to sugar molecules. These sugar molecules are used as energy source. The process by which they make sugar molecules is called photosynthesis. A very important substance in the leaf that helps in photosynthesis is chlorophyll. The chlorophyll is green in color. When the duration of sunlight and the surrounding temperature decrease following summer, chlorophyll slowly disintegrates, and the green color goes away. The bright red color is due to anthocyanins. When the stored glucose in maple leaf breakdown in the cold weather, red anthocyanins are formed. The orange color is due to carotene and yellow color from xanthophylls. These colors appear as the bright green chlorophyll vanishes from the leaves.

The following is the step-by-step description of a science fair project done by a middle school student. To cite this page, the link is http://www.sciguru.com/science-projects/ or SciGuru.com Science Projects

Hypothesis.
The change in colors of Red Maple leaves and Sweetgum leaves in the fall is due to change in level of pigments.

Aim.
To separate alcohol soluble pigments in Red Maple and Sweetgum leaves, collected during the Fall, using chromatography paper.

Materials
1 Nickel (American 5 cent coin)
3 Glass Bowls
Pestle
Teaspoon
32 Fl oz. of alcohol (70% ethanol, 30% water, you can buy Rubbing Alcohol from the nearby pharmacy)
Small pan, half full of hot water
1.5 mL V bottom tubes (from Sergent-Welsch)
Tube holder (from Sergent-Welsch)
Chromatography paper (from Sergent-Welsch)
Dropper (from Sergent-Welsch)
Rectangular prism glass container (vase from Michaels)
String
Tape
Red Maple leaves and Sweetgum leaves

Procedure.

Leaves
Leaves were gathered from a Red Maple tree and a Sweetgum tree, approximately once every two weeks from the beginning to the end of fall season. Leaves could not be collected from the Red Maple tree during the last collection as leaves were already fallen from this tree. The collected leaves were stored in the kitchen freezer in labeled airtight Ziploc bags.

Alcohol Extract
The alcohol soluble compounds in the leaves were extracted as follows. Three nickel size were cut out of the leaves from each time period. The leaf pieces were thoroughly crushed in a glass bowl with a pestle in one tsp. of alcohol.(You can use a small mortar and pestle used in the kitchen, you may obtain one from the superstore) The bowls were covered in clear plastic wrap and placed on a pan half filled with hot water for about ten minutes. The alcohol extract was transferred to a labeled 1.5 mL tube and allowed to sediment for thirty minutes. The supernatant was transferred to another tube using a dropper.

Paper Chromatography
One inch wide chromatography paper was cut into twenty centimeter long strips. A pencil line was drawn two centimeters above the bottom of the paper and the alcohol extracted samples were loaded drop by drop. Each drop was air dried before putting another drop. Seven drops of each specimen were used. A string was passed through the loop created by folding and taping the top of the paper over such that the lower edges were aligned. Alcohol was poured into the glass container at a height of 1 cm. The papers were lowered into the container with care while pulling the string taut, then the ends of the string were taped to the edges of the glass. The container was then covered with clear wrap to reduce alcohol evaporation. The chromatogram was run until the solvent front reached approximately 1 inch from the top of the paper. The paper was then taken out to dry.

Calculation of Rf Value
The distance traveled by each pigment and the solvent front were marked on each chromatogram. The Rf value was calculated using the following formula.
Rf= ( The distance the pigment traveled)/(The distance the solvent traveled)

A picture was taken of each chromatogram to document the experiment.

Results:
Alcohol soluble pigments in Red Maple and Sweetgum leaves were separated by paper chromatography. The leaves collected at different stages of color change in the Fall demonstrated different pigment patterns on the chromatogram.

Red Maple Leaves:
Leaves were collected from the Red Maple tree only four times. We saw a dramatic color change from dark green at the first collection to fully red leaves by the end of the season. The chromatogram of the green leaf showed carotene and chlorophyll a. The chromatogram from the last collection showed no trace of chlorophyll.

Sweetgum Leaves:
Leaves were collected from the Sweetgum tree five times. The first four times the leaves were green with only small red blotches. At the last collection, all the leaves had turned orange and red. The chromatogram from the first collection showed only carotene and chlorophyll a, whereas the chromatogram from the last collection showed only red anthocyanin and carotene.

Conclusion:
The decreasing level of green chlorophyll and increasing level of red anthocyanin, is the reason for change in color of Red Maple and Sweetgum leaves from green to red in the fall.



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