Separation of Colored Pigments in Leaves
INTRODUCTION
Plants have various colored pigments in their chloroplasts to help absorb the light energy from the sun. The most common
pigments are CHLOROPHYLL b, a yellow-green pigment, CHLOROPHYLL a, a blue-green pigment, XANTHOPHYLL, a yellow
pigment, and CAROTENE, a yellow-orange pigment. These pigments can be easily isolated from green plant leaves by using a
technique called PAPER CHROMATOGRAPHY. Paper chromatography is a technique used by scientists to separate molecules on
the basis of differences in solubility in specific solvents and their ability to adhere to the filter paper. Specific molecules, dissolved
in specific solvents, travel up the paper at specific rates. This rate of travel is measured by a ratio of solvent fronts known as the Rf.
Thus, molecules that are not very soluble in the solvent and/or adhere strongly to the filter paper will travel up the paper at a
slower rate than those substances that are very soluble in the solvent, and/or do not adhere well to the filter paper.
Distance a component travels up the paper (In our case, “components” are pigments)
Rf = ------------------------------------------------------------Distance the solvent travels up the paper
Dx
Rf = ---------Df
or
Thus, by comparing the values of unknown and known molecules, one can actually identify the unknown molecule. Other types of
chromatography have been developed, using substances other than paper.
STUDENT OBJECTIVES
1. Learn the principles of paper chromatography, which is a very common and very powerful tool in the laboratory.
2. Separate the pigments in spinach leaves and determine their R f values.
PRELAB QUESTIONS (Write question and answer in your lab notebooks)
1.
What is the purpose of chromatography as a laboratory tool?
2.
How may chemicals separated by paper chromatography be identified?
3.
List and describe 4 pigments that can be extracted from leaves.
MATERIALS
Leaves, test tubes and rack, parafilm with paper clip (hooked), strip of chromatography paper, quarter, chromatography solvent, ring stand,
graduated cylinder, small metric ruler, scissors, pencil, safety goggles.
SAFTEY PRECAUTIONS
Solvents may cause irritation. If you get any of the liquid materials from this lab on your skin, be sure to rinse the affected area with water.
Also, wear safety goggles at all times during this lab.
PROCEDURE
I. Preparation of the Paper Strip
1.
TRY TO MINIMIZE TOUCHING THE STRIP. IT MAY INTERFERE WITH THE CHROMATOGRAPHY. Measure 2 cm from the bottom of
the paper strip. Lightly draw a pencil line across the strip. Use scissors to cut off the two bottom corners of the strip, forming
a pointed end. [See the diagram below]
2.
Lay your leaf on top of the line you drew on your chromatography paper and roll a quarter over the leaf tracing over the
pencil line. This should leave a colored line over the pencil line. You may need to do this twice to make sure it is a thick line .
(could also use a ruler or anything with an edge to deposit the sample)
III. Running the Chromatogram
1.
Carefully pour a small amount (3-8mL) of chromatography solvent into the test tube. Make sure it will be enough to touch the
bottom of the chromatography paper when you place the paper strip into the tube. Also make sure that the solvent begins
BELOW the line where you traced the leaf extract. (rolled the quarter) [Refer to above diagram]. Do not get the solvent on
the side of the tube. If you do, let the tube dry.
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2. Hang the paper on the hook made out of the paper clip.
3. While the test tube is in the rack, gently ease the strip into the solvent at the bottom of the tube. MAKE SURE YOUR LINE
DOES NOT TOUCH THE SOLVENT. Gently cover the tube with parafilm to seal the test tube – this is very important in order
to obtain good chromatogram results.
4. Watch the solvent creep up the paper strip. Notice how the pigments are
beginning to separate. When the solvent almost reaches the hook hole,
remove the paper strip and use a pencil to mark the top of the solvent
front. DO THIS QUICKLY BECAUSE THE SOLVENT EVAPORATES.
5. Record the distance the solvent traveled from the original pigment line.
6. Record how far the center of each pigment front traveled.
7. Calculate Rf
Questions: (Answer on your lab data sheet)
1. What pigments were you able to separate in each type of leaf?
2. How far did each pigment travel? (Which went the shortest and which went the farthest?)
3. What is the purpose for each of these pigments? (how are they used by the plant?)
4. Describe why the leaves change color in autumn. What is the biological purpose for this process?
5. Describe the process of photosynthesis. How do plants use the energy from the sun to make food?
trCalculations: Show your calculations for Rf each pigment:
Distance a component travels up the paper (In our case, “components” are pigments)
Rf = ------------------------------------------------------------Distance the solvent travels up the paper
or
Dx
Rf = ---------Df
Chlorophyll aChlorophyll bXanthophyllCarotene -
C. Attach your chromatography paper to your lab notebooks!
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