Lab # _____: Separation by Chromatography
Lab skills: 1, 3, 12, 13, 17, 21, 25, 27
Background: Paper chromatography is a useful technique for separating and identifying
pigments and other molecules from cell extracts that contain a complex mixture of
substances. As a solvent moves up the special chromatography paper, it carries along any
substances dissolved in it. The pigments move at different rates because they are not all equally soluble and because
they are attracted to the fibers of the paper to different degrees.
Cellulose is the material made by plants to form cell walls and is the most important ingredient in the paper-making
process. There are several substances made by plants that create the colors that can be observed in leaves over the
course of a growing season. Beta carotene and xanthophyll can only be observed in leaves in the fall. Chlorophyll is the
primary pigment in plants and makes plants green. The pigments collect light energy and some, like carotenoids, protect
the photosynthetic systems from the damaging effects of ultraviolet light.
In this laboratory you will identify the different colors hidden in the black ink of a marker and then separate plant pigments
from spinach leaves using the separation technique known as chromatography.
Hypothesis: If certain colors consist of more than one substance, then… (tell me if you think the pigments can or can’t be
observed using chromatography.)
Procedure:
1. Fold a piece of chromatography paper in half the long
way. Cut the paper to a point at one end.
2. Draw a medium size dot with a black wet-erase marker
about 2 cm up from the point at the bottom of the paper.
3. Pour ~ 10 mL of water into the chromatography tube. The
water will act as a solvent.
4. Hang the chromatography paper in the chromatography
tube and adjust its height with a paper clip so that just the
tip of the paper is touching the water.
5. Observe what happens as the liquid travels up the paper. Record your observations with your results on a sheet of
lined paper.
6. When the solvent line is toward the top of the paper it has done its job. Remove the paper from the tube and allow it to
dry.
7. Measure the distance from the starting point to the top of each color and to the end distance to which the water moved.
Record all data in mm in a table similar to the one at the top of the next page.
Chromatography Lab
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Distance Moved by Marker Colors
Color
Distance traveled (in mm)
Rf
Total distance water moved = _________________
8. The relationship of the distance moved by a pigment to the distance moved by the solvent is a constant called the
reference front or Rf . It can be thought of as a ratio and can be calculated using the following formula:
Rf =
distance pigment migrated (mm)
distance solvent front migrated (mm)
9. The solvent front for this situation would be the distance the water moved. Calculate the Rf values for each color
observed and record them in the last column of the data table that you made following the example above. Report
your Rf calculations to three decimals places and remember that there are no units (since it is simply a ratio).
The green, blue, red, and lavender colors that came from the black ink
should help you to understand that what appears to be a single color
may in fact be a material composed of many different pigments – such
is the case with chloroplasts!
10.
You will separate plant pigments using an organic solvent. Wear eye goggles during this portion of the lab and
be sure to keep the solvent bottle tightly closed except when you are using it because it is volatile and produces
fumes that you should not breathe.
11. Pour ~10 mL of solvent into a clean chromatography tube. Cover the stock solution container with aluminum foil to
prevent vapors from spreading.
12. Cut a new piece of chromatography paper as you did
before.
13. Use a quarter to extract the pigments from spinach leaf
cells. Place a small section of leaf on the
chromatography paper in about the same location as
you made the dot with the marker before. Use the
ribbed edge of the coin to crush the leaf cells. Use a
back and forth movement exerting firm pressure
throughout.
14. Place the chromatography paper in the cylinder. Do not allow the pigment to touch the solvent.
15. When the solvent has risen to the top of the paper, remove it from the tube and immediately mark the location of the
solvent front before it evaporates.
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16. Mark the bottom of each pigment band. Measure the distance each pigment migrated from the bottom of the original
pigment line to the bottom of the separated pigment band. Record the distance that each front, including the solvent
front, moved in a data table drawn in your results section to appear like the one below. You may be able to observe
anywhere from 2 to 5 pigment bands depending on the species and age of the spinach used.
Distance Moved by Pigment Bands
Band Number
Distance (mm)
Band Color
1
2
3
4
5
Distance Solvent Front Moved = _________________
17. Report your spinach pigment Rf values in a table that looks like the one shown below.
= Rf for beta carotene (yellow to yellow-orange)
= Rf for xanthophyll (yellow)
= Rf for chlorophyll a (bright green to blue green)
= Rf for chlorophyll b (yellow green to olive green)
18. Label and staple your chromatography papers to your lab report.
Analysis:
A. How many different colors could you see separate from the black ink?
B. List the colors that appeared from the ink separation in order from top to bottom.
C. List the colors that appeared from the leaf separation in order from top to bottom.
D. Name two different separation techniques used by biologists (Hint: think about a blood sample and a DNA test).
E. Why don’t all substances possess the same patterns of colors?
F. If you used a green felt-tip marker, what results would you expect to get from chromatography?
G. Will the Rf value of a specific substance change if it comes from a different source? Why or why not?
Chromatography Lab
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