PAPER CHROMATOGRAPHY SEPARATES PLANT PIGMENTS
Objectives:
1) Prepare an extract of plant tissue (e.g., leaves, flowers).
2) Apply the technique of paper chromatography as a method for separating individual plant
pigments contained in plant tissue extracts containing pigment blends.
2) Describe the application of this technique to the study of plant pigments and develop related
testable questions.
3) Generate ideas about ways to improve the technique to yield better results.
INTRODUCTION TO PLANT PIGMENTS
A "pigment" is simply a molecule that absorbs and reflects light. Recall that white light actually
consists of many colors – you may have learned “ROY G BIV” in high school physics as a way to
remember the colors of light that make up the white light of the “visible spectrum”. Different
pigments appear different colors because they have differing abilities to absorb and reflect
various colors of light. (A more thorough discussion of the light-absorbing properties of
pigments will be presented in the Spectrophotometry lab.) The broad array of colors found in
plant tissues such as leaves, flowers, and fruits, can be accounted for by the presence of
literally thousands of different kinds of plant pigments.
Through plant breeding and horticultural practices, humans have manipulated plants’ pigment
producing capabilities to serve our own desires. News was made recently when a true blue rose
cultivar was successfully created in Japan. In nature, color is an important attribute of plants
that serves to attract pollinators to receptive flowers and signal fruit ripeness to seed
dispersers. In some instances, colors may also serve to warn potential predators of poisonous
or toxic substances contained in plant tissues.
Color-producing pigments have other important roles in plants beyond regulating interactions
with animals. Chlorophyll is a pigment that reflects green light, but absorbs red and blue
wavelengths and is critical for the light reactions of photosynthesis. Flavonoids are an
important class of plant pigments that block ultraviolet (UV) radiation that can damage cell
proteins and DNA. Many flavonoids, including anthocyanins (a subcategory of flavonoids) have a
role in the chemical defense of plants as they are toxic to many herbivores and pathogens –
especially insects and fungi.
As you may know from the popular media, there is currently a substantial research effort in
place to explore the potential health benefits of plant pigments to humans. In popular
literature, these plant-based compounds are often collectively referred to as “phytochemicals”;
most are also pigments. Flavonoids, anthocyanins, and carotenoids are just some of the
categories of plant pigments known to have antioxidant properties. “Antioxidant” is a general
term used to describe any substance that has the ability to neutralize “free radicals” which
cause cellular damage by removing electrons from surrounding molecules. Many lines of research
suggest that consuming a diet rich in plant pigments may slow the process of cellular aging and
reduce the risks of some types of disease, such as cancer, heart disease, and stroke. Cosmetic
companies are even jumping on the “antioxidant bandwagon” by adding seductive blends of
antioxidant-rich “botanical extracts” to their shampoos, makeups, and lotions in the hopes of
prolonging our youthful glow!
A few categories of pigments are listed below along with their characteristic range of colors.
Pigment Type
Anthocyanins (subclass of flavonoids)
Anthoxanthins (subclass of flavonoids)
Betacyanins
Carotenoids
Chlorophylls
Xanthophylls (a subclass of carotenoids)
Colors
blue/purple/red
yellow - ivory
yellow - red/purple
yellow - red
greens
ivory - yellow
Some plant pigments you may be familiar with that are of current interest in nutritional and
pharmaceutical research are listed below, though there are many more!
Pigment
anthocyanins
Color
blue/purple/red
beta-carotene
orange/yellow
curcumin
yellow
Found in
berries, grapes, red peppers, beets, eggplant,
plums
carrots, pumpkin, sweet potatoes, citrus, papaya,
melon, squash
turmeric
lutein
lycopene
yellow/orange
red
kale, broccoli, spinach
tomatoes, watermelon, red grapefruits
zeaxanthin
yellow
corn
PAPER CHROMATOGRAPHY METHOD
Step 1: Prepare chromatography papers. Cut the chromatography paper into strips following
dimensions suggested by your instructor. Draw a fine pencil line across (but not all the way
across) the strip about 2-3 cm from one end - this is the "origin". Handle the papers by the
edges, taking care to touch them as little as possible - oils from fingertips can interfere with
the migration of pigments up the paper.
the “solvent front” is the position of the liquid solvent on the
chromatography paper at any given time. the solvent will
gradually move from the bottom toward the top of the paper,
carrying dissolved pigments with it. stop the chromatogram
before the solvent front reaches the top of the paper and mark
the location. you will use this distance to calculate Rf
mark the location of each pigment at the time the chromatogram is
stopped
origin (where you apply the pigment extract)
Step 2: Prepare your plant extract. In this procedure, we are interested in the “qualitative”
assessment of the presence of individual pigments, we are not quantifying them (determining
how much of a pigment is present). For this reason, exact measurements of plant tissue and
extracting solvent are not necessary. (In the Spectrophotometry lab, we will be quantifying
plant pigments, so careful measurements will be necessary.).
Place the plant tissue of interest into a mortar (a few leaves or flowers). Add a small amount of
ethanol to the plant tissue and grind completely with a pestle to release the pigments into
solution. Continue adding ethanol (or plant tissue) as necessary to create a few milliliters of
very dark extracted liquid. Your goal is to create a highly concentrated solution but avoid a
paste-like consistency.
Step 3: Load the extract onto the chromatogram. Dip a capillary tube into the liquid portion of
your extract. (Your extract may contain fragments of plant tissue which will clog the capillary
tube. To minimize this, tilt your mortar slightly to allow the liquid fraction to run away from
the solids.) Allow the extract to migrate up the capillary tube. Dab the end of the capillary onto
the origin of your chromatography paper. The extract will move out of the capillary tube onto
the paper as it is absorbed into the paper fibers.
Allow the extract to dry completely on the paper, then repeat with another load of extract. In
order to concentrate the pigments on the paper, you will need to apply several loads of extract
(probably 4 - 6). After each loading, wait until the paper is fully dry before applying the next
load. Depending on the type of sample you have and the amount of water in it, it may take
several minutes for the sample to dry (2 - 10 minutes). You may use a hairdryer on the
lowest/coolest setting to speed the process. Do not proceed to step 3 until your sample is
completely dry.
Don’t forget: you will need to prepare 1 chromatogram for each chromatography solvent you will
be testing. Your instructor will let you know which chromatography solvents should be tested
for a given plant tissue. Use the same extract to prepare all the chromatograms.
Step 3: Set the chromatogram in the chromatography solvent. Place your paper strip into the
solvent container provided with the origin end down. Make sure that the level of the solvent is
below the origin on your chromatogram – you do not want to submerge the origin in the solvent.
Check the chromatogram frequently to observe the movement of solvent and pigment up the
chromatography paper.
WARNING: The petroleum ether/acetone solvent is highly flammable and can be dangerous if
inhaled. Take care to avoid inhaling the fumes as much as possible and keep clear of any flame,
spark, or other ignition source!!
Step 4: Stop the chromatogram and record your results. When the solvent "front" is within 2-3
cm from the top of the paper, remove the chromatogram. Use a pencil to quickly mark the
location of the solvent front. Allow the chromatogram to air dry, then trace and label the
pigments you observe (they will fade over time).
Step 5: Identify your pigments. Calculate the Rf value (described below) for each pigment in
each chromatography solvent tested. Consult your instructor (or reference provided) about the
identity of the pigments you isolated – record all your data in your notebook.
IDENTIFYING PIGMENTS ISOLATED BY PAPER CHROMATOGRAPHY
Different pigments have different sizes, shapes, and physical properties (e.g., different
solubilities in our chosen solvent). As a result, different pigments will move at different rates
up the chromatography paper allowing them to visibly separate from one another. Once the
pigments are separated, they can be identified by a variety of methods.
One way to determine the identity of a pigment is to physically remove it from the paper and
assay it by another method. For example, we could elute (remove) a pigment from the
chromatography paper by dissolving it in another solvent, such as ethanol and measuring its
absorption spectrum using the spectrophotometer. The resultant spectrum could be compared
to the known spectra for different pigments by searching in an appropriate reference manual.
An alternative method is to calculate the “Rf value”, a ratio representing the distance a pigment
travels relative to the distance the chromatography solvent travels. Again, we can then match
the Rf value and color of our unknown pigment to known values recorded in a reference manual.
Rf value = distance from origin to pigment
distance from origin to solvent front
Remember – because pigments vary in their solubility in different solvents, the Rf value for a
given pigment is tied to the chromatography solvent. In other words, chlorophyll b will have an
Rf value in petroleum ether/acetone that is different from its Rf value in BAW.
POSTLAB QUESTIONS:
1. Sketch each resulting chromatogram into your lab notebook (or scan it/take digital photos
and paste it in) and construct an associated table that summarizes your data. Give each table a
title that identifies the plant and chromatography solvent used. Include in your table: the
number of pigments isolated with each chromatography solvent (e.g., “pigment 1”, “pigment 2”,
etc. . .), a color description of each pigment, its Rf value, and its likely identity.
2. What was the initial color of the plant extract(s) you used? What is the relationship
between the colors of the pigments you isolated and the color of the original extract?
3. Why is it important to stop the chromatogram before the solvent front reaches the top of
your chromatography paper? What would happen to your chromatogram if you let it run too
long?
4. In some cases, two or more pigments may overlap each other on the chromatogram, making it
difficult to isolate and identify them. What are some things you could do to modify the method
to improve pigment separation (distance between pigments)?