Chromatography lab
Background: All of life on Earth depends on the process of photosynthesis.
Photosynthesis is a process that plants, cyanobacteria, , and algae use to convert carbon
dioxide and water, in the presence of light energy, into a sugar called glucose.
Photosynthesis requires the presence of special pigments that can absorb the energy of
light. A pigment is a substance that absorbs light of a particular wavelength. Its color
depends upon the color of light that it reflects. For example, a green substance appears
green because it reflects green light and absorbs all other colors of light, especially red
and blue.
The most important plant pigments in photosynthesis are the “chlorophylls.” Green
plants contain both chlorophyll a (bright green) and chlorophyll b (yellow-green). In
addition to chlorophyll, the leaves of many green plants also contain one or more other
pigments, including “carotenes,” which are faint yellow, “xanthophylls,” which are
yellow, and “anthocyanins,” which are red. The presence of these other pigments is
masked by the abundance of chlorophyll during most of the year.
The pigments in plant cells can be separated from one another by a technique known as
chromatography. Chromatography is a technique for separating and identifying
substances in a mixture, based upon their solubility in a solvent. It is one of the most
valuable techniques chemists and biochemists use to determine ingredients that give
flavor or scent, analyze environmental pollutants, identify drugs in urine, and even
separate proteins that can identify evolutionary relationship.
When a dye mixture is placed on a strip of chromatography paper and placed into a
solvent solution, the individual substances in the mixture will migrate up the
chromatography paper at different rates. The rate of migration is based upon the
absorption capacity of the chromatography paper and the solubility of the sample in the
solvent. As the solvent moves up the chromatography paper strip substances in the
mixture that are soluble in the solvent are carried along up the strip. On the other hand,
the substances that are more attracted to the chromatography paper than to the solvent
stop moving and form bands or spots along the paper strip. After chromatography, each
band or spot on the chromatogram can be assigned a Relative Mobility Factor (Rf) which
is characteristic of the specific substance that is associate with it. The Rf is a ratio of the
distance a substance travels to the distance the solvent travels. It is calculated as follows:
Rf = Distance solute traveled (Dunknown)
Distance solvent traveled (Dsolvent)
The Rf value for a particular substance is constant with a consistent set of
chromatographic conditions. Therefore, substances can be identified according to their
Rf value when compared to a known standard.
Purpose: In this lab, students will perform chromatography to separate the pigments
present in a spinach leaf using various solvents, and will compare the effects of each
solvent used to the pigments shown. Students will also calculate the Rf values for each
pigment.
Materials:
Goggles
Chromatography paper
Scissors
Pencil
Quarter
Spinach
Glass vials with lids
Solvent (acetone, isopropyl alcohol of varying concentrations)
Ruler
Tweezers
Procedure:
1. Obtain a piece of chromatograph paper from your teacher.
2. Cut the bottom to a point by making two cuts at a 45 degree angle.
3. About 1 cm up from the bottom, draw a line in pencil. Use a ruler to make it even.
4. Place a leaf of spinach on top of the line. Apply the pigment to the paper using a
quarter, rolling over the spinach along the line 5-6 times.
5. Pour a small amount of the assigned solvent (your teacher will assign each group a
solvent) into the glass vial.
6. Carefully place your chromatography paper into the vial. MAKE SURE YOU DO
NOT LET THE PIGMENT SUBMERGE INTO THE SOLVENT!!! This will ruin your
experiment as the pigment will just dissolve into the solvent rather than migrate up the
paper. Then, place the cap on the tube.
7. Watch the movement of the solvent up the paper for two (2) minutes. You should see
the pigment begin to move with it. Set this aside for another 18 minutes.
8. After 20 minutes, remove the paper from the chamber and IMMEDIATELY mark the
height of the solvent front (the furthest that the solvent ran up the paper).
9. You should have several colors on the paper. Gently lay the chromatogram on a
paper towel. Now find the different pigments on the paper. To calculate the Rf of
each pigment, circle the area where the same color exists. After you have made each
circle, find the midpoint of each circle and draw a line across.
10. Take a ruler and measure the distance to the solvent front. This is your total distance
traveled by solvent. Measure each midpoint for each pigment and calculate the Rf for
each and place in the table on your data sheet.
11. Answer the questions that follow.
Data:
Solvent:
Distance solvent traveled:
Line
Color
Probable
pigment
Distance solute
traveled
Rf value
1
2
3
4
5
Analysis questions:
1. How many pigments were you able to identify on your chromatogram?
2.
Compare your results with other groups. Did they get the same number of
pigments as you? If not, what was different?
3.
Again, comparing your results with other groups, did they get the same Rf values
as you?
4.
What could account for any differences between the Rf values between groups?
5.
Which of these pigments was the most soluble in the solvent? The least? How
do you know?
6.
Many trees have leaves that are green in the summer and red, yellow, or orange
in the fall. If these pigments are present in the leaf, why don’t they appear until
the fall?