Plant Pigments (Micah Hagan)

***Clarification to the demonstration***

-- In the demo I stated that chlorophyll b is the blue green band and chlorophyll a is the lowest green band. The opposite is correct. Chlorophyll a is the blue green band and chlorophyll b is the lowest green band.

Spinach chromatography (Micah Hagan)

High School Biology

Principles covered: This activity uses paper chromatography to separate and identify the various pigments found in plant cells.

Objective: Students will set up a chromatography experiment in order to separate individual pigments from a spinach leaf. Students will calculate Rf values for each pigment.

Standards Covered

Biology

1. The fundamental life processes of plants and animals depend on a variety of chemical reactions that occur in specialized areas of the organism’s cells. As a basis for understanding this concept:

f. Students know usable energy is captured from sunlight by chloroplasts and is stored through the synthesis of sugar from carbon dioxide.

Investigation and Experimentation

1. Scientific progress is made by asking meaningful questions and conducting careful investigations. As a basis for understanding this concept and addressing the content in the other four strands, students should develop their own questions and perform investigations. Students will:

a. Select and use appropriate tools and technology (such as computer-linked probes, spreadsheets, and graphing calculators) to perform tests, collect data, analyze relationships, and display data.

BACKGROUND

If asked to define a plant, many students start by keying in on their relatively consistent color: “They’re green.” Indeed, the importance of the chlorophyll that gives plants their green color can’t easily be overstated. As a result of the characteristics of these pigments, plants have the ability, through a harvesting of the sun’s energy, to produce their own food. They are autotrophs. Ultimately, this process, photosynthesis, is the source of food for the other life on earth as well. On some level, we are all dependent on photosynthesis. Good thing we know a lot about it.

Photosynthesis is a series of light-driven reactions that converts carbon dioxide and water into energy-rich sugars. The process begins when pigments absorb light energy from the sun. Chlorophyll a is the most important pigment in photosynthesis, and absorbs light in the blue and red zones of the visible light spectrum. This pigment reflects green light and plants appear green because we see that reflected light. When the chlorophyll a molecules absorb light photons, they channel that energy and excite electrons that begin a series of reactions to create chemical energy. Accessory pigments, like chlorophyll b, carotenoids, and xanthophyll help optimize the harvesting of light energy, as they absorb wavelengths of the visible spectrum not absorbed by chlorophyll a.

Because chlorophyll a is much more abundant than any of the accessory pigments, it tends to mask their presence in the plant. This lab uses paper chromatography to separate the pigments present in a spinach leaf. When pigments are transferred to chromatography paper and submerged in the proper solvent, the solvent moves through the pigments and up the paper. Each respective pigment differs in molecular size, polarity, and solubility, and so move up the paper at different speeds and varying distances.

Materials

1. Chromatography paper

2. Solvent (Petroleum ether)

3. Spinach

4. Pencil

5. Ruler

6. Paper clip

7. Graduated cylinder

8. Parafilm

PROCEDURE

1. Make a faint pencil mark across a strip of chromatography paper approximately 2cm from the end of the paper. Make sure you handle the paper by its edges so that oil on your fingers does not contaminate the paper.

2. Place a leaf of spinach along the pencil line. Use a dime to roll along the line, in the process transferring pigment extract to the chromatography paper. Let dry completely and repeat 3-5 times.

3. Straighten a paper clip and bend one end through the top of the chromatography paper. Bend the other end over the top of the graduated cylinder so that the bottom of the chromatography paper is suspended just above the bottom of the tube (about at the 2-5 ml mark).

4. Remove the paper and pipette in chromatography solvent to just above where the end of the paper was in the last step. Caution: Solvent is extremely flammable and should not be inhaled. Use only in well ventilated areas.

5. Place the paper strip in the cylinder so that the end nearest the pigment is submerged in the solvent, but the 2cm line (pigment extract line) is above the solvent level.

6. Immediately cover the cylinder paper, with parafilm. Wait and watch for 10 minutes.

7. Remove the paper and properly discard of the solvent.

8. Mark the position of the solvent front (the full distance the solvent travelled up the paper) with a pencil and set the strip aside to dry.

9. Observe the bands of color and draw your chromatography strip in the results section below. Four bands should appear: a yellow band of carotenes, a yellow band of xanthophyll, a blue-green band of chlorophyll a, and a yellow-green band of chlorophyll b.

10. Measure the distance from the pigment origin to the solvent front and from the pigment origin to each pigment band. Calculate the Rf for each pigment. Rf is the distance traveled by the pigment divided by the distance traveled by the solvent front (from the pigment’s origin). Rf values differ depending on the type of solvent used, but the order should remain the same: yellow-colored carotenes travel the farthest, followed by yellow xanthophyll, blue-green chlorophyll a, and then the yellow-green chlorophyll b.

Data, Results, and Analysis

- See attached lab

Real World Applications

- Fall color change in leaves can be explained by this lab. During the winter, many plants rest and utilize stored energy. During this time, chlorophyll disappears from tissues and the accessory pigments are more readily seen.

- While disgusting, carrots contain lots of carotenoids, which are good for eyesight in young students.

- Plant pigments have been used for thousands of years as dyes.

References

http://en.wikipedia.org/wiki/Biological_pigment#Commercial_uses

http://www.huntington.org/huntingtonlibrary.aspx?id=694&linkidentifier=id&itemid=694