Chemistry 103
Lab 1: Adaptations for Photosynthesis
Objective: To investigate photosynthesis and the chemicals involved by thin layer
chromatography (TLC).
Introduction: Photosynthesis generally refers to synthesis of glucose from light,
carbon dioxide and water. Glucose is a critical molecule for energy production in
cellular metabolism and consequently photosynthesis is a technique for
harnessing light to produce cellular energy.
TLC of Spinach
Although chlorophyll is the most well know pigment contained in plants it
certainly isn’t the only one present. Below are the structures and description of
chlorophylls and other components that are in the pigments.
The colored pigments from a plant fall into 2 categories: chlorophylls and
carotenoids. Carotenoids are yellow pigments that are involved in the
photosynthesis process.
They include xanthophylls, which are oxygencontaining 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b except that in each case the magnesium ion (Mg2+) has been replaced by two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 plate you will see in the following in order of decreasing R f 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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The most widely used methods of separating components of organic chemical
mixtures involve some form of chromatography. The most modern method of
separating mixtures in organic chemistry is chromatography. Chromatography
is defined as the separation of a mixture of two or more different compounds by
distribution between two phases, one of which is stationary and the other
moving. The method depends on the different solubilities, or adsorptivities, of
the substances to be separated relative to the two phases between which they are
to be partitioned.
In thin layer chromatography (TLC) the ‘stationary’ phase is the adsorbent silica,
which is bound to an aluminum-backed plate (also called a TLC plate). Silica is
considered a polar substance since the surface of the crystals consists of polar
hydroxyl (OH) groups. The ‘moving’ phase is an organic solvent system that, by
capillary action, will move up the stationary silica coated plate. All solvent
systems will be considered non-polar relative to the silica adsorbent.
The sample mixture is usually applied as a small spot near the base of the TLC
plate (called ‘spotting’). The plate is then put into a solvent reservoir where, by
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capillary action, the solvent will rise up the plate. As the solvent ascends the
plate, the compounds in the sample are partitioned between the moving liquid
solvent and the stationary solid phase. This process is called developing the TLC
plate.
When developing a TLC plate, the various components in the mixture are
separated. This separation is based upon each compound’s distribution
equilibrium between the solvent and adsorbent. See the figure below:
Compound Distribution Equilibrium
Each compound will have a unique distribution equilibrium depending mainly
upon the polarity of the compound (based on intermolecular forces between the
compounds being separated and the adsorbent). An example is that of a polar
compound vs. a non-polar compound. The distribution equilibrium of a polar
compound will favor the adsorbent since the adsorbent is highly polar (“like
dissolves/attracts like”). The non-polar compound however, will have less
affinity for the polar adsorbent and will have an equilibrium favoring solubility
in the mobile solvent. The consequence of this is that polar compounds will
‘stick’ to the stationary TLC plate while non-polar compounds will separate and
travel upward with the solvent. When developing a TLC plate we can state that
each compound in the mixture will ascend the plate at a different rate; polar
compounds ascend slowly, less polar compounds ascend quickly.
In this experiment you will isolate a mixture of colored chlorophylls (see pages 12 for structures) from spinach leaves and then separate this mixture into its
individual components using TLC.
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In thin layer chromatography (TLC), the stationary phase is adsorbent silica
bound to a thin flexible plastic sheet, called a TLC plate. Silica (silicon dioxide) is
considered to be a polar substance. The mobile phase is an organic solvent
system (in other words, a mixture of one or more organic solvents), which, by
capillary action, will move up the stationary phase. Solvent systems are
considered to be non-polar compared to the silica, though there are degrees of
non-polarity.
The sample mixture is applied as a small spot near one edge of the TLC plate;
this is called “spotting”. The plate is then put vertically into a solvent system
reservoir such that the spotted edge is placed down (but keeping the spots above
the level of the reservoir) and the solvent system will ascend the plate. As the
solvent system goes up, the compounds in the sample mixture will (ideally)
separate; some of the compounds should stick to the stationary phase and some
should dissolve and be carried up the plate along with the mobile phase. This
process is called “developing” the TLC plate.
To get an idea of why compounds in the mixture separate, consider a mixture
that contains both a polar and a non-polar compound. The polar compound will
favor the adsorbent silica (the stationary phase) because the silica is highly polar
(following the rule of “like dissolves like”). The non-polar compound will favor
dissolving in the non-polar solvent system and travel upward with the solvent
front. Thus, each compound will ascend the plate at different rates, with more
polar compounds tending not to rise quickly. Separation is achieved!
Pre-Lab Questions:
1. What role does light serve during photosynthesis?
2. Why are CO2 and H2O necessary?
3. List the toxic chemicals we will be using.
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Materials:
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Spinach leaves
Acetone, hexane, 70/30 hexane/acetone
Anhydrous sodium sulfate
Mortar and pestle, 2 centrifuge tubes, Pasteur pipets
Hot plate, beakers, test tubes
TLC plates, micro capillaries, filter paper, watch glass cover (or
aluminum foil), pencil
Test tubes
Procedure
A. Isolation of Pigments:
1. Weigh about 0.5 g of fresh spinach leaves. Cut or tear the spinach leaves
into small pieces and place them in a mortar along with 1 mL of acetone.
Grind with a pestle until the spinach leaves have been broken into
particles too small to be seen clearly. If too much acetone has evaporated,
you may need to add an additional portion of acetone (0.5-1.0 mL) to
perform the following step.
2. Using a Pasteur pipet, transfer the mixture to a centrifuge tube. Rinse the
mortar and pestle with 1.0 mL of acetone and transfer the remaining
mixture to the centrifuge tube. Centrifuge the mixture (be sure to balance
the centrifuge). Using a Pasteur pipet, transfer the liquid to a centrifuge
tube with a tight fitting cap.
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3. Add 2.0 mL of hexane to the tube, cap the tube, and shake with mixture
thoroughly. Then, add 2.0 mL of water and shake thoroughly with
occasional venting. It’s important to thoroughly dissolve the pigments in
the hexane before adding the water. Centrifuge the mixture to break the
emulsion, which usually appears as a cloudy, green layer in the middle of
the mixture.
4. Remove the bottom aqueous layer with a Pasteur pipet. Using a Pasteur
pipet, prepare a column containing anhydrous sodium sulfate to dry the
remaining hexane layer, which contains the dissolved pigments (see the
figure on next page). Gently place a small plug of cotton into a Pasteur
pipet and tap it into position using a glass rod. Add about 0.5 g of
sodium sulfate and tap the column with your finger to pack the material.
5. Clamp the column in a vertical position and place a dry test tube under
the bottom of the column. With a Pasteur pipet, transfer the hexane layer
to the column. When all the solution has drained, add 0.5 mL hexane to
the column to extract all the pigments from the drying agent. Evaporate
the solvent by placing the test tube in a warm water bath and directing a
stream of air into the vial. Once evaporated, dissolve the residue in 7-10
drops of hexane.
Column for Drying Extract
TLC Development Chamber
B. TLC of spinach extract:
1.
Obtain two 1-inch TLC plates and micro capillaries from your instructor.
These plates have a flexible backing, but they should not be bent
excessively. They should be handled carefully or the adsorbent may flake
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off them. Also, they should be handled only by the edges; the surface
should not be touched.
2.
Using a lead pencil (not a pen) lightly draw a line across the plates (short
dimension) about 1 cm from the bottom (on the coated side). At the center
of this line make a light mark. This is the point at which the spinach
extract will be spotted.
3.
To spot the TLC plate, fill the capillary tube by dipping one end into the
spinach extract. Capillary action fills the pipet. Empty the pipet by
touching it lightly to the thin-layer plate at the mark that is at the center
of the 1 cm line from the bottom (The spot must be high enough so that it
does not dissolve in the developing solvent).
4.
When the pipet touches the plate, solution is transferred to the plate as a
small spot. The spot should be no larger then 2 mm in diameter and
should be a fairly dark green. If you do not have a dark green spot, you
may spot again using another sample of your spinach extract.
5.
Allow the solvent to evaporate completely between successive
applications, and spot the plate in exactly the same position each time. It
is important that the spots be made as small as possible and that the
plates not be overloaded.
6.
When the first plate has been spotted it is ready to be placed in a development
chamber. For a development chamber you will use your large beaker lined with a
piece of filter paper and cover (see the figure above). When the development
chamber has been prepared, obtain a small amount of the development
solvent (70/30 mixture of hexane/acetone). Fill the chamber with the
development solvent to a depth of about 1/4 inch (about 10 mL of
solvent). Be sure that the liner is saturated with the solvent. The solvent
level must not be above the spots on the plate or the samples will dissolve
off the plate into the reservoir instead of developing.
7.
Place the spotted plate in the chamber and allow the plate to develop (the
solvent will slowly move up the TLC plates). Since the backing on the
TLC plates is very thin, if they touch the filter paper liner of the
development chamber at any point, solvent will begin to diffuse onto the
adsorbent surface at that point.
8.
When the solvent has risen to a level about 1 cm from the top of the plate,
remove the plate from the chamber and, using a lead pencil, mark the
position of the solvent front. Let the plate dry. Lightly outline all the
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observed spots with a pencil. Before proceeding, make a sketch of the
plate in your notebook and label each spot by color. Using a ruler
marked in millimeters, measure the distance that each spot has traveled
relative to the solvent front.
9.
Under an established set of such conditions, a given compound always
travels a fixed distance relative to the distance of the solvent front. This
ratio of the distance the compound travels to the distance the solvent
travels is called the Rf value. This can be expressed as a decimal fraction:
Rf = distance traveled by substance/distance traveled by solvent front
(see figure below). Calculate Rf values for each observed spot.
10. Repeat the above TLC of you spinach extract but use a different
proportion of hexane/acetone for your developing solvent (be sure to
record the proportion used in your notebook). Once this plate has
developed, sketch the plate in your notebook, label the spots by color and
calculate Rf values for all the spots as described above.
Rf Values
Post-Lab Questions
1. Most carbohydrates have the general formula CXH2XOX. What is the waste
product of producing glucose from CO2 and water? Why is this significant?
2. Summarize your results (number of spots, colors, Rf values), note any
interesting observations and make any possible conclusions about the
experiment (successful vs. unsuccessful and reasons why).
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3. Discuss how changing the ratio of acetone:hexane might change R f values of
your pigments – for example, if you increased the amount of acetone in your
second trial, would you expect a more polar pigment to have a higher or lower Rf
than it did in the first trial?
4. What role do the various pigments (other than chlorophylls) serve in
photosynthesis?
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