Extraction and Fractionation of Plant Pigments from Spinach Leaves
When we see a green leaf from a plant what we actually see is light reflected off
the surface of the leaf, specifically, the green wavelengths of light. During Autumn, or
when leaves begin to die, we see different colors from the same leaves. Actually, leaves
contain many different pigments – the compounds that absorb and reflect different
wavelengths of light. In lab today we will extract the pigments from leaves of spinach,
then separate the different pigments that are present using a process called paper
chromatography. Then, based on the separation of pigments, we will try to distinguish
exactly which pigments are present.
A few hints for today’s lab:
The solvent we will use to separate the pigments is petroleum ether:acetone. This
solvent is very hydrophobic, so any pigments that are very “greasy” or
hydrophobic themselves will interact with the solvent (move with it). The more
hydrophilic pigments will not react well with the solvents, but will react well with
the chromatography paper (not move well with the solvent). Handle all the
solvents carefully.
The chromatography paper itself is very polar. It is made of cellulose, which
contains sugars with –OH groups that form hydrogen bonds with oxygen atoms of
the pigments.
EXTRACTION OF PIGMENTS
1. Obtain about 2-3 nice dark green spinach leaves (or coleus, or beet leaves). Remove
and discard the major veins of the leaf and save the rest of the tissue.
2. Use a razor or scissors to cut the leaf into tiny pieces. Add the pieces to a chilled
mortar and pour about 15ml of ice-cold acetone onto the leaf pieces. Add a pinch of
sand to the mortar. You will find these materials in the freezer.
3. Grind this mixture with the pestle for about a minute.
4. Pour the ground-up contents of the mortar into a 50 ml Erlenmeyer flask, label the
flask, and affix the stopper.
5. Swirl the flask vigorously for 30 seconds or so, then place the flask into the
refrigerator for 10 minutes. Allow the pulp to settle.
6. While you are waiting, prepare the paper and jars for the rest of the experiment as
described below. (Step 1 of PAPER CHROMATOGRAPHY)
7. Retrieve your flask from the fridge. Don’t disturb the pulp at the bottom of the
flask.
PAPER CHROMATOGRAPHY
1. Use the large jars as chromatography tanks. Place a small piece of filter paper into the
jar so that the paper rests against the wall of the jar. This small piece of paper will help
saturate the atmosphere of the jar with vapors from the solvent. Gently pour petroleum
ether:acetone solvent into the jar to a depth of about 1 cm. Cover the jar tightly with foil.
2. Obtain a long sheet of chromatography paper. Do not handle the paper with your
fingers. Oils from your skin will alter how the solvent reacts with the paper and affect
your results. One member of your team should wear a pair of latex gloves to handle the
paper.
3. Use a fine pencil to mark a thin light pencil line across the wide dimension of the
paper 2cm from the bottom. Your pigment extract will be applied along this pencil line.
4. Dip the end of a glass capillary tube into the pigment solution in the flask and quickly
streak the capillary tube across the paper along the pencil line. Leave about a 1cm side
margin on the edges of the paper where no pigment is applied. Your instructor will
demonstrate this technique for you.
Allow the solvent to dry completely before reapplying more extract.
5. Repeat streaking the paper with extract until you have a dark green line on your paper.
This may take 6-10 applications. Take your time – the narrower your line of pigment
extract the better results you will get.
6. Let the paper dry completely, then curl it into a tube and staple the edges at each end
to form a cylinder. Do not handle with your fingers, and do not overlap the ends
when you staple them together. Make sure the pigment line is at the bottom end of the
cylinder, and is visible.
7. Get your jar, remove the small strip of paper, and place your cylinder into the jar, with
the pigment end of the tube in the solvent. Cover tightly with foil right away. Do not
move the jar once the paper has been placed into the solvent.
8. Allow the pigments to separate for 10-15 minutes, or until there is a clean
separation of four to five bands of different pigments. Let the solvent run until it is
about 1cm from the top of the paper. Do not let the solvent run off the top of the
paper. When the solvent has reached 1cm from the top remove the paper from the jar
and immediately mark the solvent front with a pencil. Allow the paper to dry
completely.
9. Once dry, delineate each of the colored bands with a pencil, and record the color and
the distance each band moved from the starting line or origin (the pencil line you drew at
the beginning). Describe the colors as specifically as you can (olive green, grass green,
yellow orange, etc). Record the distance of the solvent front from the origin.
10. Calculate the Rf values for each of your pigments
Rf = distance traveled by pigment / distance traveled by solvent
DATA ANALYSIS
For each of your pigments you have an Rf value and a description of the color of
each band. Pigments may be identified by their color and the Rf values.
Chlorophyll a is blue green
Chlorophyll b is olive-green
Beta-carotene is yellow-orange
Violoxanthin is yellow
Lutein is yellow
(Violoxanthin and lutein are both xanthophylls pigments and might overlap)
1. Use your chromatogram to fill in the following table:
Pigment / solvent band
Distance
traveled (cm)
Rf
2. What do the Rf values indicate about the relative attraction of the pigments to the more
polar paper or the more non-polar (hydrophobic) solvent? Review the introduction to this
lab if necessary…
3. From your chromatogram, what is the order in which the pigments separated?
Chlorophyll a
Chlorophyll b
Beta-carotene
Lutein
Violoxanthin
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4. Is each band one pure pigment? Explain.
HAND IN THIS SHEET AT THE END OF LAB WITH YOUR CHROMATOGRAM.
ONE SHEET PER GROUP.
NAMES
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