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 isnt 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
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except that in each case the magnesium ion (Mg 2+) has been replaced
by two hydrogen ions.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 (bluegreen), Chlorophyll b (green), Xanthophylls (up to 3 spots, yellow)

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 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 compounds
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 1-2 for structures) from spinach leaves and then separate
this mixture into its individual components using TLC.
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.

Materials:

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.
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. Its 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.

ColumnforDryingExtractTLCDevelopmentChamber
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 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.

Whenthefirstplatehasbeenspotteditisreadytobeplacedinadevelopment
chamber.Foradevelopmentchamberyouwilluseyourlargebeakerlinedwitha
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 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 R f 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.

RfValues

Post-Lab Questions
1. Most carbohydrates have the general formula C XH2XOX. What is the waste
product of producing glucose from CO2 and water? Why is this significant?
2. Summarize your results (number of spots, colors, R f values), note any
interesting observations and make any possible conclusions about the
experiment (successful vs. unsuccessful and reasons why).
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 R f
than it did in the first trial?
4. What role do the various pigments (other than chlorophylls) serve in
photosynthesis?

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