Name: __________________________________
Block: ________
Date:_______________
Bio A: Paper Chromatography – Chloroplast & Pigment Analysis (35 points)
Introduction
When you look at a leaf, the green pigment chlorophyll is usually the only pigment that appears to be
present. Actually, chlorophyll is only one of many types of pigments present in the leaf and one of several that
are involved in the process of photosynthesis. Once removed from the leaf, the photosynthetic pigments can be
separated from one another and identified using a process called chromatography.
Theory of paper chromatography
A small sample of a mixture is placed on porous paper which is in contact with a solvent. The solvent
moves through the paper due to capillary action and dissolves the mixture spot. The components of the sample
start to move along the paper at the same rate as the solvent.
Components of the mixture with a stronger attraction to the paper (stationary phase) than to the solvent
will move more slowly that the components with a strong attraction to the solvent (mobile phase). The
difference in the rates with which the components travel along the paper, over time, leads to their separation.
Particular mixtures will have chromatographic patterns that are consistent and reproducible as long as the paper,
solvent, and time are constant. The more polar something is the further it will travel up the chromatography
paper. This makes paper chromatography a qualitative method for identifying some of the components in a
mixture.
Materials
Test tube
Penny
Water
Spinach leaves
Chromatography paper
Acetone (solvent)
Ruler
Pencil
Markers
Parafilm
Procedure:
Part 1 – Spinach leaf
1. Obtain a spinach leaf, a penny, a test tube rack, test tubes, and a piece of chromatography paper (just
long enough to fit from top to bottom of the jar).
2. Cut the tip of the paper such that it forms a point of a triangle.
3. Draw a line across the paper 1 cm up from the triangle. This is your “start line”.
4. Place your spinach leaf on top of the chromatography paper line that was drawn. Roll your penny above
the spot over the line to smash the pigment on top of the line.
5. Pour approx. 1 cm of chromatography solvent (acetone) into the chromatography jar.
6. Place the chromatography paper with the pigment into a test tube so the tip of the triangle dips into the
solvent (acetone). Do not submerge pigment lines below the solvent level. Obtain a piece of parafilm
and tightly cover the test tube. Place it into the test tube rack.
7. Allow the solvent (acetone) to rise for about 15 minutes or until the solvent line nears the top of your
papers.
8. Some possible colors and the pigments they represent are:
o Faint yellow – carotene
o Yellow – xanthophyll
o Bright green – chlorophyll a
o Yellow-green – chlorophyll b
o Red – anthocyanin
9. Measure the distance from the start point to the front line and each of the pigment lines. Record these
measurements in the data table. Calculate the Rf values for each pigment according to the following
formula:
Part 2 – Marker colors
1. Perform the same task except with a scented black marker instead of a spinach leaf. Place a dot with
scented black marker on the drawn pencil line. Use water instead of acetone.
2. Repeat this again for another color marker of your choice. Use water instead of acetone.
(5 points)
1. Draw the piece of chromatography paper when it is done below (3 drawings): (6 points)
Spinach Leaf
Black Sharpie Marker
Other Color Marker
2. Describe the results of the chromatography tests in words for each of the 3 tests. (3 points)
Spinach Leaf
Black Sharpie Marker
Other Color Marker
Analysis & Conclusions:
1. What is chromatography? What does it tell you? (3 points)
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
2. What pigments did you see in the spinach leaf? How did you know? (3 points)
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
3. Which of these pigments from the spinach leaf would be considered the most polar? Which one would be the
least polar? How do you know? (3 points)
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
4. Explain 1 use for 3 different pigments that would be found in a plant. (3 points)
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
5. What would happen if the plant was missing its carotenoids? Why would that occur? [Hint: Think about what
the job of those pigments is] (3 points)
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
6. Many trees have leaves that are green in the summer and red, yellow, or orange in autumn. Where were these
colors during the summer? Why did they suddenly appear in autumn? (3 points)
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
7. Explain 1 similarity and 2 differences between your three results (spinach leaf, black sharpie and other
colored sharpie). (3 points)
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
__________________________________________________________________________________________
Chlorophyll Fluorescence
INTRODUCTION
When a pigment absorbs light, electrons of certain atoms in the pigment molecules
are boosted to a higher energy level. The energy of an absorbed and a photon is
converted to the potential energy of the electron that has been raised to an excited
state. In most pigments, the excited electron drops back to its ground-state, or
normal orbit, and releases the excess energy as heat. Some pigments,
including chlorophyll, emit light as well as heat after absorbing photons.
In the chloroplast, these excited electrons jump from the chlorophyll molecule to a
protein molecule in the thylakoid membrane, and are replaced by electrons from the
splitting of water. The energy thus transferred, is used in carbohydrate production.
This release of light is called fluorescence. Chlorophyll will fluoresce in the red part
of the spectrum, and also give off heat. In this lab, you will observe this fluorescence
by separating the chlorophyll from the thylakoid membrane.
MATERIALS
Spinach leaves
Flashlight or small lab light
Mortar and pestle
Test tube
Acetone
Filter paper
25-mL graduated cylinder
Funnel
Ring stand or funnel rack
Safety goggles
PROCEDURE
1. Grind the spinach leaves using a mortar and pestle.
2. Add acetone to the ground leaves, using enough acetone and spinach leaves to get
between 10 and 15 mL of extract.
3. Set up your filtering apparatus, and using proper filtering technique, filter the
extract to a test tube. NOTE: Use a small amount of acetone to wet the filter paper,
to hold it into place, instead of water.
4. Shine a flashlight, or other similar light source, through the test tube and extract.
5. Observe the fluorescence of the chlorophyll at a 90 degree angle to the
flashlight.
Analysis Questions:
1. What color is chlorophyll when you shine a light on it? Why would it be that color?
2. Why do you need to grind up the spinach leaves for this experiment?
3. This chlorophyll is involved in what important plant process?
4. If the chlorophyll were to be removed from these leaves, what possible colors could be shown? Why would
this occur?