Name:
8.2A
Date:
Period:
Chromatography
Introduction: What gives leaves their color? Why do they change colors in the fall?
The answers to these questions revolve around light and pigments.
Energy from the sun travels to Earth in the form of light. Sunlight, which our eyes perceive as
“white” light, is actually a mixture of different wavelengths. Many of these wavelengths are
visible to our eyes and make up what is known as the visible spectrum. Our eyes see the
different wavelengths of the visible spectrum as different colors: shades of red, orange,
yellow, green, blue, indigo, and violet.
Plants gather the sun's energy with light-absorbing
molecules called pigments. Photosynthetic
organisms capture energy from sunlight with
pigments. The plants’ principal pigment is
chlorophyll. The two types of chlorophyll found in
plants, chlorophyll a and chlorophyll b, absorb light
very well in the blue-violet and red regions of the
visible spectrum. However, chlorophyll does not
absorb light well in the green region of the
spectrum, as shown in Figure 8–4.
Figure 8-4
Leaves reflect green light, which is why plants look green. Plants also contain red and
orange pigments such as carotene that absorb light in other regions of the spectrum.
Most of the time, the intense green color of chlorophyll overwhelms the accessory
pigments, so we don’t notice them. As temperatures drop late in the year, however,
chlorophyll molecules break down first, leaving the reds and oranges of the accessory
pigments for all to see. The beautiful colors of fall in some parts of the country are
the result of this process.
Recall from studying the cell that plants and other photo-synthetic eukaryotes, photosynthesis takes
place inside organelles called chloroplasts. Chloroplasts contain an abundance of saclike
photosynthetic membranes called thylakoids (THY luh koydz). Thylakoids are interconnected and
arranged in stacks known as grana (singular: granum). Pigments such as chlorophyll are located in
the thylakoid membranes. The fluid portion of the chloroplast, outside of the thylakoids, is known
as the stroma. The structure of a typical chloroplast is shown in Figure 8–5.
Figure 8-5 Sketch and TEM of a Chloroplast
Prelab Questions:
1. What are the different wavelengths of light? ______________________________________
___________________________________________________________________________
2. How do plants absorb sunlight? _________________________________________________
___________________________________________________________________________
3. Why don’t we see all pigments all the time in leaves? _______________________________
___________________________________________________________________________
4. Describe the chloroplast’s structure. _____________________________________________
___________________________________________________________________________
Purpose: To separate and identify the different pigments located in leaves.
Materials: (Per pair of students)
2 Plant leaves
2 Stoppers
Test tube holder
Solvent
Chromatography paper
Coin
2 Test tubes
2 Tacks
Colored pencils
Procedure:
1. Obtain two test tubes with tight fitting corks, and thumbtacks.
Label one test tube “spinach” and the other “lettuce”.
2. Obtain two pre-cut chromatography paper strips.
3. With a pencil, NOT A PEN, on both paper strips - draw a line 2
cm from the end. Label the top of one strip with an “S” and the
other with a “L” (for spinach and lettuce).
4. Strip “S” – lay the spinach leaf upside down over the pencil line.
Using a coin, gently roll across the leaf over the line. If you
pressed hard enough to damage the leaf you should now see a
green line. Repeat the coin rolling until you have a nice dark
green line. Keep the line as thin as possible – you do not want a
green splotch, you want a green line. When your line is dark
green, put aside the strip. Do not get it wet. Discard leaf.
5. Strip “L” – repeat procedure as with strip “S”. This time use the
lettuce instead of spinach leaf.
6. Tack the top of the chromatography strips into the stoppers. Tack them appropriately so the
bottom of the strip is near the bottom of the test tube (strip should not “crunch” up when
stopper placed on test tube).
7. Call instructor for solvent.
8. Allow solvent to soak into strip for 10 minutes undisturbed.
9. While waiting, record the appearance of the spinach and campus leaves on the next page.
10. After 10 minutes, remove stopper and un-tack strips. Solvent will be returned to instructor.
11. Sketch in color your observations of the two strips.
Data Collection:
“S” Spinach Leaf Color ________________
“L” Lettuce Leaf Color ________________
Data Table 1 Spinach and Lettuce Leaf Chromatography Results
“S” Spinach Chromatography Strip
“L” Lettuce Leaf Strip
In Data Table 1, label the pigments observed for each strip using the following information.
Pigment
Chlorophyll a
Color
Observed
Bright Green
Chlorophyll b
Dull green
Carotenoid
Xanthophyll
Anthocyanin
Orange, Red
Yellow
Blue, Purple
In Data Table 2, show work for calculating Rf values for each pigment seen
Rf Vaule is the distance travelled by the sample divided by the distance travelled by the solvent front in
chromatography.
Rf Vaules
Chlorophyll a
Chlorophyll b
Carotenoid
Xanthophyll
Anthocyanin
Spinach
Lettuce
Analysis Questions:
1. What are pigments and what do they do for plants? ________________________________
___________________________________________________________________________
___________________________________________________________________________
2. Different pigments can be observed through chromatography but not when observing the
leaf itself. Why is that? _______________________________________________________
___________________________________________________________________________
3. Describe the color of the two leaves and compare/contrast their pigments present.
Leaf Color
Pigments Present
Spinach Leaf
Leaf Color
Pigments Present
lettuce Leaf
4. Why would you expect more pigments in the spinach leaf? ___________________________
___________________________________________________________________________
___________________________________________________________________________
5. Which pigment has the largest molecular weight? How do you know? __________________
___________________________________________________________________________
___________________________________________________________________________
6. Which pigment has the smallest molecular weight? How do you know__________________
___________________________________________________________________________
___________________________________________________________________________
7. What is the purpose of chromatography? _________________________________________
___________________________________________________________________________
___________________________________________________________________________
8. Record the Rf values that another student in class calculated for their strip. Are they the
same or are they different? ____________________________________________________
___________________________________________________________________________
___________________________________________________________________________
9. If the Rf values were similar, explain why you think that is true. If the Rf values were
different, explain why you think this is true. _______________________________________
___________________________________________________________________________
___________________________________________________________________________