Lab 3: Extraction of Algae Pigments and Thin Layer Chromatography (TLC)
Objectives:
- To learn the highly useful thin layer chromatography technique.
- To study the chemical composition of plant leaves.
Introduction:
One of the most modern methods of separating mixtures in chemistry is
chromatography. Chromatography utilizes a mobile phase and a stationary phases.
Much as the name implies, the mobile phase is a solvent that flows sample and the
stationary phase (also known as the adsorbent). The stationary phase is generally a
solid powdery material with a polar surface. Silica (SiO2) and alumina (Al2O3) are the
two most common stationary phase materials.
In thin layer chromatography (TLC) the stationary phase is the adsorbent silica,
which is bound to an plastic-backed plate called a TLC plate. Silica is considered a
polar substance since the surface of the crystals consists of polar hydroxyl (OH) groups.
The mobile phase is an organic solvent mixture that, by capillary action, will move up
the stationary silica coated plate. The polar is relatively non-polar compared to the
silica adsorbent.
The sample mixture is usually applied as a small spot near the base of the TLC
plate (this process is called spotting). The plate is then put into a solvent reservoir
where, by capillary action, the solvent will rise up the plate. As the solvent ascends the
plate, the compounds in sample will cling to the mobile phase and dissolve into the
mobile phase. This process is called developing the TLC plate.
When developing a TLC plate, the various components in the mixture are
separated based upon polarity. Polar molecules will tend to spend a greater amount of
time clinging the polar stationary phase than nonpolar molecules. The more strongly
molecules cling the stationary phase, the slower the mobile phase will transport them
across the place. Thus polar molecules tend to move across the plate more slowly than
nonpolar molecules.
Compound Distribution Equilibrium
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In this experiment you will extract a mixture of colored molecules from algae
powder and then separate this mixture into its individual components using TLC.
Pre-Lab: (Answers submitted at the beginning of lab)
1. Summary of the procedure in your own words.
2. Read the short article “The Chemistry of Autumn Colors” and write a 1-2
paragraph summary of this article. Article can also be found at this web address:
http://scifun.chem.wisc.edu/chemweek/fallcolr/fallcolr.html
Procedure
1. On a balance weigh 0.5 grams of algae powder and 0.5 grams of anhydrous magnesium
sulfate.
2. Transfer the powder to a large test tube and add 2.0 ml of acetone. Stopper the test tube and
shake vigorously for approximately one minute. You need to make sure that the solvent and solid
are well mixed. Rinse sides of test tube with 1 - 2 ml acetone, and allow the mixture to stand for
10 minutes.
3. Use a pipette to carefully transfer the solvent above the solid (should be green) into a small
test tube. Do not suck the solid into the pipette. Cover the tube to avoid evaporation. Do not
discard this solution because it will be needed again in step 10.
4. Obtain a TLC chamber (a glass jar with a cover) and add developing solvent ( a mixture of pet
ether, acetone, cyclohexane, ethyl acetate and methanol). The solvent should completely cover
the bottom of the chamber to a depth of approximately 0.5 cm or 1/8”.
5. Obtain a TLC plate (a silica gel coated plastic sheet) which has been precut and make a dot
with a pencil on the matte powder-coated side approximately 1.0 cm from the bottom of the strip.
Do not use pen because the ink will run when exposed to the solvent.
6. Fill a capillary tube (the very small glass tubes) by placing it in the extract. Apply the extract
to the center of the dot on the matte powder-coated side of the TLC plate by quickly touching the
end of the TLC applicator to the plate. You want the spot to be approximately 0.5 cm or 1/8”
across. Allow to dry. Repeat several times to make a concentrated dot of extract (your instructor
will demonstrate this process). Be sure to let the spot dry between applications and before you
place it in the chamber.
7. Carefully place the TLC plate in the TLC chamber. The TLC plate should sit on the
bottom of the chamber and be in contact with the solvent (solvent surface must be
below the extract dot). Screw the lid on the TLC chamber.
8. Allow the TLC plate to develop (separation of pigments) until the solvent is close to the top of
the place (roughly 1 cm away). As the solvent moves up the TLC plate you should see the
different colored pigments separating.
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TLC Development Chamber
9. Remove the TLC plate from the chamber when the solvent is approximately 1.0 cm from the
top of the TLC plate. With a pencil, mark the level of the solvent front (highest level the solvent
moves up the TLC plate) as soon as you remove the strip from the chamber. Gently mark the
location of all visible spots on the TLC plate with a pencil because they will fade over time.
10. Use the solution to prepare a TLC plate for each member of the group.
11. Calculate and record the Rf values (see below).
Rf Values: Rf = distance traveled by substance/distance traveled by solvent front. Calculate Rf
values for each observed spot. Note that this example has two spots but the spinach should show
3 to 6 spots.
Waste Disposal:
Put all extra organic solvent into the organic waste bottle in the hood. Algae
residue can go into the waste beaker in the hood.
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Lab Report Guide:
- 1. Results (5 pts)
o Create a table containing following information about each spot
 Appearance
 Distance traveled
 Rf value
o Sketch your TLC plate
o Attempt to identify each of the spots using the below information:
The colored pigments from a plant fall into 2 categories, Chlorophylls and Carotenoids.
Caroteniods are yellow pigments that are involved in the photosynthesis process.
Xanthophylls are oxygen-containing carotenes (OH and C=O) The carotenes are shown
below. The Green pigments are the Chlorophylls that act as the principal photoreceptor
molecules of plants. There are two different forms, chlorophyll a and chlorophyll b.
The two forms are identical except that the methyl group that is shaded in the structural
formula of Chlorophyll a is replaced by an aldehyde (C=O) group in chlorophyll b.
Pheophytin a and pheophytin b are identical to chlorophyll a and b except that in each
case the magnesium ion (Mg2+) has been replaced by two hydrogen ions.
On your TLC place you will see in the following in order of decreasing Rf value (top of
plate to bottom); Carotenes (yellow), Pheophytin a (grayish), Pheophytine b (grey, may
not be visible), Chlorophyll a (blue-green), Chlorophyll b (green), Xanthophylls (up to 3
spots, yellow)
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2. Post Lab Questions (5 pts)
o Typed answers to the Post Lab questions. Note that single sentence
answers will not suffice. State the answer to the question followed by a brief
description of the evidence supporting that answer.
Post-Lab Questions:
1. Why are the chlorophylls less mobile than the carotenes on the TLC plate?
2. Is it possible to have an Rf value greater than 1? Explain.
3. There are a number of differently colored molecules present in the spinach.
Explain what role these molecules might serve in photosynthesis.
4. You may have noticed that many of the spots on the TLC faded upon exposure to
air. Does the speed with which the spots faded suggest anything about their
strength as antioxidants? You may wish to research antioxidants before
answering this question.
5. Very polar compounds are sometime purified by reverse phase chromatography
where the stationary phase is very nonpolar while the solvent is very polar. Why
might one want to use this technique with extremely polar compounds?
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