BIOL 0129 PHOTOSYNTHETIC PIGMENTS INTRODUCTION

BIOL 0129 PHOTOSYNTHETIC PIGMENTS

INTRODUCTION

The photosynthetic pigments are responsible for absorbing and trapping light
energy in the early steps of photosynthesis. Before coming to lab develop a hypothesis
relating to pigments and light (i.e. why are there several pigments in green leaves?).
What benefits do the plants get by having several pigments? Also, consider what
happens to leaves on deciduous trees in the Northern Hemisphere in the fall and pose a
hypothesis to explain this phenomenon. Which of the hypotheses developed can be
tested using this experiment? Why? Be sure to include these hypotheses in the
introduction of your lab report.

The major pigments of photosynthesis are the chlorophylls. The two chlorophylls
found in green plants are chlorophyll a (chl a) and chlorophyll b (chl b). Certain other
chlorophylls (chlorophyll c and bacteriochlorophylls) are found in non-green algae,
protistans, and photosynthetic bacteria. Other pigments include carotenoids and
phycobilins, sometimes referred to as the accessory pigments. Carotenoids occur in all
photosynthetic organisms, while phycobilins occur in the red algae and cyanobacteria.

In this experiment, the photosynthetic pigments from spinach leaves will be
extracted and separated using the technique of paper chromatography. After separating
the pigments, their absorption spectra will be obtained using a spectrophotometer.

Chlorophylls

The chlorophylls have a similar molecular structure. Each has a porphyrin ring and
a long phytol side chain. Although the porphyrin ring resembles the prosthetic group of
hemoglobin and cytochrome, it has a central magnesium atom instead of iron. The
alternating double and single bonds of the porphyrin ring make chlorophyll an efficient
light-absorbing molecule and determine the general shape of the absorption spectrum.
The phytol chain, which is almost devoid of double bonds, contributes little to the
absorption spectrum. See drawings on pg. 7. As is the case for other compounds, the
specific absorption maxima of any chlorophyll depends on the solvent in which it is
dissolved.

Carotenoids

There are two classes of carotenoids, the carotenes and the carotenols. All
carotenoids have long isoprenoid chains, with alternating double and single bonds.
Structurally, the carotenes are composed entirely of carbon and hydrogen, whereas the
carotenols also contain oxygen in the form of hydroxyl or keto groups. See drawings on
pg. 7.

Paper Chromatography

Chromatography is a technique used to separate the components of a mixture.
There are various types of chromatography (column, paper, thin-layer, gas), but in all
cases the separation is achieved by distribution of components between a fixed or
stationary phase and a moving or mobile phase. In paper chromatography, the
components of a mixture are separable into discrete zones on a sheet of filter paper.

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The mixture is initially spotted or streaked near one end of the paper. If the
separated substances are to be extracted later for further analyses, the procedure is
called preparative paper chromatography. With a capillary tube, the mixture is streaked
on the chromatography paper: enough sample is applied so that there will be an
adequate amount for subsequent extraction and spectrophotometric analysis.

For ascending paper chromatography, the appropriate solvent is added to the
bottom of a chromatography jar. The atmosphere in the jar should be saturated with
solvent vapor prior to adding the paper. The paper is placed in the jar so that the streak is
above the level of the solvent. Then, the solvent moves up the paper by capillary action,
past the sample, toward the end of the paper.

During this process, termed development, the solutes separate and form a trail of
discrete bands on the chromatogram. Separation of the components is usually measured
by the Rf value. The Rf value is given by the equation:

Distance traveled by the solute
Rf =

Distance traveled by the solvent from the origin

For the numerator, the distance is measured from the origin either to the center or
to the leading edge of each spot or band. The denominator is the distance from the origin
to the solvent front. The Rf values can be used to identify the various solutes when the
experimental conditions are very carefully controlled.

Analysis of Spinach Pigments

The photosynthetic pigments are extracted from spinach by grinding the leaves in
acetone. The paper is then streaked with the spinach extract and suspended in a
chromatography jar previously equilibrated with vapors of the solvent, a 9:1 mixture of
petroleum ether and acetone.

When separation is completed, identify the pigment bands by their colors and
relative positions on the chromatogram. The major pigments appear in 5 bands: in order,
from the origin to the solvent front, they are chl b(olive-green), chl a (blue-green),
violaxanthin (yellow), lutein (yellow), and β-carotene (yellow-orange).

Each pigment or pigment group will be eluted from the chromatogram by cutting
out each band and soaking the strips of paper in acetone. Violaxanthin and lutein, the
carotenols, will be combined and treated as a single group. The absorption spectrum of
each pigment or pigment group will then be determined.

You will also perform a quantitative analysis for the two major pigments, chl a and
chl b. The absorption coefficients (α) for chl a and chl b in 80% acetone are: for chl a, α
663 = 82.04, and for chl b, α 645 = 45.60. Using the Beer-Lambert equation, A = α⋅c⋅l, you

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will determine the concentration (mg/ml) of each chlorophyll in a dilution of the eluted
sample. Finally, you will calculate the ratio (chl a)/ (chl b), a value that is characteristic for
each plant species.

PROCEDURES

Preparation of the Chromatography Chamber

The solvent mixture is extremely flammable so that the chromatography should be
carried out in a fume hood. Before handling the paper, hands should be washed and
thoroughly dried. The paper should be handled as little as possible and only on the
edges.

1. Obtain a piece of chromatography paper. Cut it to 19X19 cm.

2. Add the solvent mixture to the chromatography chamber at least 30 min. before
adding the streaked chromatography paper so that the atmosphere in the chamber will
become saturated with solvent vapor.

Add freshly prepared solvent (9 vols. petroleum ether*:1 vol. acetone, mixed well) to a
height of approximately 2 cm and cover the chamber with a lid that is sealed at the rim
with Vaseline. Be careful not to get any solvent on the Vaseline.

*CAUTION: Petroleum ether and acetone are extremely flammable and should be kept
away from heat, sparks, or an open flame.

Preparation of the leaf extract for the whole class

1. Weigh out 15 g of fresh spinach leaves after removing major veins. Cut into small
pieces.

2. Place into blender; add 60 ml 100% acetone and homogenize.

3. Filter first through cheese cloth and then through Whatman filter paper in a Buchner
filter under vacuum.

4. Place filtrate in foil-covered 150 ml flask, cap, and put on ice.

Preparative Paper Chromatography

1. With a pencil and ruler, draw a light line across the width of the chromatography paper,
about 3 cm from the bottom. This will insure that the extract, which will be streaked on
this line, is not be immersed in the solvent. Make a light mark on each end of the line
about 1 cm from the edge of the paper.

2. Using a capillary tube, make 10 streak applications of the pigment extract along the
line between the two marks. Do not let the pigment touch the edge of the paper. The

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capillary tube is filled by immersing the tip in the extract. Hold the tube at a 45o angle and
draw it along the pencil line. Move your arm quickly so the pigment does not form a large
spot at the beginning of each streak.

Streak each successive application in the direction opposite of the preceding one to
insure an even line at the end of the ten applications. Allow each application to air dry
before making the next. The final thickness of the streak should be no more than 6-7
mm.

3. Use two paper clips and thread to place the streaked chromatography paper in the
equilibrated chromatography chamber, being careful not to get any Vaseline on the
paper. Allow the chromatogram to develop in the dark or in very dim light for 45-60 min.
or until there is clean separation of the 5 bands. Stop the development before the solvent
front reaches the end of the paper.

4. Remove the chromatogram from the chamber and put a pencil mark at the leading
edge of the solvent front before it dries. Hang it in the hood to dry. Record the distance
traveled by the solvent front and by the leading edge of each band. Also record the color
of each band and what pigment it contains. This must be done before elution.

Elution and Spectrophotmetry

1. Allow the spectrophotometer to warm up for at least 5 min. Set the wavelength at 400
nm.

2. Label 4 cuvettes as follows: chl b, chl a, carotenols, β-carotene.

3. Cut out each of the 5 bands on the chromatogram; cut them into thin strips and place
them in the appropriate cuvette (cut them long enough so you can remove them from the
cuvettes but not so long that they extend out of the acetone); place the two carotenol
bands, violaxanthin and lutein, in the same cuvette. Thus, you will be eluting 4 pigments
or pigment groups: chl b, chl a, carotenols, and β -carotene.

4. Add 5.0 ml acetone to each cuvette, cover each tube with parafilm, and allow the
pigments to elute for 5 min., occasionally swirling each tube. Invert each cuvette twice to
mix the contents, then remove the paper strips with forceps, draining the paper against
the side of the tube.

5. Prepare a fifth cuvette with a dilute solution of the original extract (0.2 ml of extract +
4.3 ml acetone). Invert twice to mix the solution.

Also prepare a reference blank with 100% acetone.

6. Measure the absorbance of each sample at 20 nm intervals starting at 400 nm and
proceeding to 700 nm. The spectrophotometer must be adjusted with the reference blank
prior to the absorbance readings at each wavelength. All 5 cuvettes can be read with the

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one adjustment for the blank at each wavelength. Move the filter lever to the right before
you do the 600 nm reading.

7. To determine the concentration of chl a and chl b, dilute the solvent to 80% acetone as
follows:

Transfer 4 ml of the chl a eluate and 4 ml of the chl b eluate to two clean cuvettes. To
each add 1 ml distilled water, cover, and invert each tube twice to mix the contents. Also
prepare a reference blank with 80% acetone.

8. Measure the absorbance of the chl a solution (in 80% acetone) at 663 nm and the
absorbance of the chl b solution (in 80% acetone) at 645 nm. Be sure to adjust with the
reference blank at each wavelength.

RESULTS AND DISCUSSION

The following should be included in the results and/or discussion of the lab report.
Incorporate them in the appropriate section. Do not write the results and
discussion as a list of answers to the questions.

1. Calculate the Rf value for each pigment.

2. What do the Rf values indicate about the relative solubilities of the pigments in the
solvent? What would be the order of solubility if the pigments were separated in water
instead of the nonpolar solvent mixture (petroleum ether and acetone).

3. Explain the relative solubilities of chl b and chl a in the solvent on the basis of their
molecular structures.

4. Explain the relative solubilities of the three carotenoids in the solvent on the basis of
their molecular structures.

5. Using Excel, plot wavelength (ordinate, x axis) versus absorbance (abscissa, y axis )
for chl b, chl a, carotenols, β-carotene, and the diluted extract. Include a title, axes
labels, and legends. Place all pigment values on the same graph. When printing the
graph choose the landscape choice for paper orientation and make the graph fill 75% of
the page.

6. What is the wavelength of the absorption maximum (or maxima) for each pigment or
pigment group? Compare your results to references.

7. Why does a plant use several pigments instead of one or two? Why are plant leaves
green?

8. From the absorbance readings at 663 nm and 645 nm and the Beer-Lambert equation,
determine the concentration of chl a and chl b in the diluted eluates.

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(chl a) = mg/ml; (chl b) = mg/ml.

Show the calculations in the results.

Beer-Lambert Equation:

A = α⋅c⋅l A = absorbance

α = absorption coefficient
c = concentration
l = length of light path (l=1)

9. What is the ratio (chl a)/(chl b) in spinach?

10. Do the results support the hypothesis in the lab introduction? Why or why not?