SBI 4U
Identifying Plant Pigments by Spectrophotometry
Introduction:
Photosynthetic organisms use chlorophyll pigment to harness energy from photons and
convert it into chemical energy. Only certain wavelengths of visible
light can be absorbed by the pigment and in this investigation, we will identify the
primary wavelengths and produce and absorption spectrum for chlorophyll and other
pigments found in spinach.
Background Information: Read sections on p. 151-153 about absorption spectra.
Answer questions p. 154 # 3, 6, 8. Read the section on p. 186 about the
spectrophotometer.
Materials:
spinach leaves
mortar and pestle
acetone
microcentrifuge
scissors
test tubes and rack
sand
spectrophotometer
Procedure:
Extracting Photosynthetic Pigments
1. Weigh out 5-6 gm. of leaf material. Use only the leaf blades, not the stems.
Dispose of all other materials.
2. Cut the leaf blade into pieces (use scissors) and place them in a mortar.
3. Add 1-2 gm. of fine sand and 8 ml. of acetone.
4. Grind the materials thoroughly (5 min.)
5. Add 8 ml. of acetone and grind for one more minute. Allow the mixture to
stand for 4 min. The sand ruptures the cells, aiding in the release of pigments
that dissolve in the acetone.
6. Centrifuge the solution and decant into a new clean test tube. Retain a small
sample for the paper chromatography in part II
7. Make a 1:30 dilution of your pigment extract using acetone as a solvent. Cap
the tube to prevent evaporation.
8. Determine the absorption spectrum of your pigment extract by using the
spectrophotometer as indicated in the following steps.
a. Turn on the spectrophotometer and familiarize yourself with the position of
the control knobs. Set the wavelength control at 400nm.
b. Adjust the meter to 0% transmittance with the Power Switch/Zero control
knob (on the front left side of the instrument).
c. Obtain two sample tubes and clean them if necessary. Fill one tube with
acetone (solvent) and use it as a blank. Fill the other with the 1:30 dilution of
your pigment extract.
d. Insert the blank tube containing acetone. Align the guide mark on the cell with
the guide mark of the sample compartment and close the lid. Adjust the meter
with the Transmittance/Absorbance control so that it reads 100% transmittance.
e. Remove the blank tube and recheck the 0% transmittance. If it is necessary to
readjust, repeat step d. Insert the tube containing your pigment extract 1:30
diluted.
f. Read and record the transmittance in table 7.1. i. Change the wavelength to
425nm and repeat the adjustments in steps d to h. Continue these measurements
at 25nm intervals up to 750nm. Remember to recalibrate (steps d to g) at each
new wavelength.
g. To calculate the estimated percent absorbance, subtract the percent
transmittance from 100 percent.
Wavelength (nm)
400
425
450
475
500
525
550
575
600
625
650
% Transmittance
%Absorbance
SBI 4U
Identifying Plant Pigments by Chromatography
Submit this package with graph and answers stapled at the back - no title page
Source:
Nelson Biology 12 and University of Pittsburgh at Bradford, Science in
Motion Biology Lab 012
Introduction:
Paper chromatography is a method of separating the components of a
mixture, in this case the mixture of pigments found in concentrated plant
pigment extract. A small amount of sample is applied (spotted) near the bottom
of the paper (stationary phase) and the paper is placed in the mobile phase
(solvent). This solvent is drawn up by capillary action. Separation occurs as
each component, being different in chemical and physical composition, interacts
with the stationary and mobile phases to a different degree creating the
individual bands on the plate. The retention factor, Rf value, is used to
characterize and compare components of various samples.
Rf value = distance from origin to component spot
distance from origin to solvent front
The pigments in vegetables, flowers and leaves can be separated and
identified by using thin-layer chromatography. Green pigments, known as
chlorophylls, serve as the main photoreceptor molecules of plants. Carotenoids,
yellow pigments, aid the plant in the photosynthesis process. In addition,
xanthrophylls are contained in the chloroplasts which can be isolated and
identified using chromatographic techniques.
No two chromatograms are identical. Several factors influence the
reproducibility of chromatograms.
1. humidity: time of application and during developing
2. solvents: contaminated solvents will cause results to vary
3. developing chamber: atmosphere inside chambers must be the same
spotting technique: inconsistent spotting results in variations
Pigments can also be identified by their absorbance curves using a
spectrophotometer.
Pigments in concentrated chlorophyll
Pigments in concentrated carotenoid
Pigment
Visible Color
Rf
Pigment
Visible Color
Rf
Carotene
Yellow
0.98
Alpha
Yellow-orange
0.97
Carotene
Xanthophyll
Yellow
0.86
Beta Carotene
Yellow-orange
0.94
Xanthophyll
Red
0.8
Lycopene
Yellow-orange-
0.81
red
Phaeophytin a
Dark grey
0.67
Leutein
Yellow-brown
0.75
Phaeophytin b
Light grey
0.6
Violaxathin
Yellow-brown
0.66
Xanthophyll
Yellow
0.5
Neoxathin
Yellow-brown
0.28
Chlorophyll a1
Light blue-green
0.48
Chlorophyll a
Dark blue-green
0.46
Chlorophyll b1
Light yellow-green
0.30
Chlorophyll b
Dark yellow-green
0.25
Xanthophyll
yellow
0.15
Purpose:
Identify plant pigments by chromatography.
Materials:
spinach leaves
mortar and pestle
acetone
microcentrifuge
scissors
test tubes and rack
chromatography paper
sand
Procedure:
Chromatography
1. p. 184, 185 (Extraction of Pigments)
capillary tube
paper clip
graduated cylinder
chromatography solvent
(90% petroleum ether
10% acetone)
aluminum foil
spectrophotometer
2.
p. 185 Chromatography
3.
Straighten a paper clip and reshape it to form a hook at each
end. The hook is going to hang off the edge of the graduated
cylinder instead of from a cork.
4.
Place 10-20 ml of the solvent into the test tube.
5.
Place the filter paper strip on the bench. Using the
microtubule, daub a small drop of the spinach extract in the
centre of a line drawn across the tapered end of the filter paper.
6.
Allow this spot to dry.
7.
Repeat the daubing process several times until the spot is dark
green, or at least the colour of your extract. Dry after each
application.
8.
Hang the filter paper strip in the graduated cylinder so that the
tapered end is in the solvent and the extract spot is above the
solvent. Cover the top with aluminum foil.
9.
Allow the solvent to rise until it is within 2 cm of the top of the
strip. Remove the strip from the test tube.
10. Quickly mark the position of the solvent front and each of the
pigment bands with a pencil and allow the strip to dry
Observations:
Draw a diagram of the chromatography strip using coloured
pencils to represent the separated pigments.
Spinach Pigment
Chromatography
Band #
(from tip)
1
2
3
4
Colour
Rf (ratio of fronts)
Pigment
Discussion Questions
1. Spectrophotometer
a)
b)
c)
d)
e)
f)
g)
At which wavelength in the range 400-650 nm does the pigment extract
absorb the most light? b) Construct a line graph of the data with percent
absorbance along the vertical axis and wavelength along the horizontal
axis. Indicate the colours of the visible spectrum corresponding to the
wavelength along the horizontal axis.
Which pigments are responsible for the spikes (highest points) in the
graph?
Which pigments of an intact spinach leaf would be least visible? Why?
What colour is absorbed the least by the pigment extract?
Compare the graph to the absorption spectrum in Figure 11 on page 152.
How does your graph compare to this one?
Identify the pigments in your extract using figure 14 on p. 153.
Fill out your chromatography chart and compare your findings to the
absorption graph from the spectrophotometer. Identify the similarities
and discrepancies between the types of pigments found using these two
methods. Which is more reliable and why.