CAPIRAL, Mikaella G.

MT 2-6
Chem 001: Problem Solving Investigation in Analytical Chemistry
PROBLEM
Are red flowers really red?
Two students were having an argument about
pigments in red flower. One said that red petals
can only contain the red color, whereas the other
students thought that the red color could actually
be a mixture of color. Plan an experiment to
investigate if the pigment in red flowers consists
of one or more colors. In your investigation think
about how you can extract the pigment and
check whether it consists of one or more color.

In your notebook, assign a number to each

different type of flower, starting with the number
1 and going up. Using a pencil, number three
strips "1," three other strips "2," and three other
strips "3" at the top of the strip, as shown in
Figure 1. This is so that you can identify which
flower was used with which strip later. (If you are
investigating more than three different types of
flowers, similarly continue to number the test
strips.)

PLAN FOR INVESTIGATION

A. Purpose of the Investigation
The purpose of this investigation is to know
whether the pigment in red flowers consist one or
more colors. The goal of this experiment is to
analyze the pigments found in flowers using
paper chromatography.
B. Materials/ Apparatus
The materials used in this experiment include
filter paper, scissors, pencil, ruler, clean jar or
drinking glass, large- mouth glass jar, water (at
least 60 mL, distilled water is preferable, but tap
water is also suitable), 70% isopropyl alcohol,
measuring cup, piece of scratch paper, coin, timer
or clock, and of course red flower petals. You will
need at least 2 flower petals from at least 3
different plants. Tip: Larger petals work better
than smaller petals.
C. Steps/ Procedure
Cut the chromatography filter paper into strips
that are about 2 cm wide and as long as the
large-mouth glass is tall (the strips should all be
the same size). You will want to make at least
three strips for each flower you want to
investigate, or a minimum total of nine strips.
Use a ruler and pencil to draw a line across each
paper strip, horizontally, 2 cm from the bottom,
as shown in Figure 1. This is the origin line,
where the sample will later be spotted. Tip: Do
not use a pen for writing on the strips because
the ink will run when the solvent passes through
the strips.

Figure 1. Chromatography strip with a pigment sample.

In your notebook, make a data table that lists the

kinds of flowers you will be testing. Also make
four columns titled something similar to "Color of
the Pigment Band," "Distance to the Solvent
Front (in cm)," "Distance to the Top of the Band
(in cm)," and "Retention Factor (Rf)."
In a clean jar, drinking glass, or mug, dilute the
70% isopropyl rubbing alcohol in half with water
by mixing 60 mL (1/4 cup) of water with 60 mL
of isopropyl alcohol. This water and isopropyl
alcohol mixture is your solvent. Pour a small
amount of the solvent into your large-mouth
glass jar, about 2 cm deep.
Next, transfer the pigments from one type of
flower onto a strip of chromatography paper. Take
one of the paper strips you prepared with this
flower's number and place it on top of a piece of
scratch paper on a hard surface. Note: Some
pigments can stain so the paper strip should be
prepared on a piece of scratch paper to protect
the surface beneath it from getting stained.
Lay a petal from the selected flower on the paper
strip, over the origin line. Roll a coin, like a

wheel, over the petal and across the origin line.

Push down hard so that the petal is crushed and
a strip of pigment is visibly transferred to the
strip (See Figure 2). Repeat this for about three
to four times (using a fresh, unused part of the
petal each time) so that a thick line of pigment is
transferred to the strip, on the origin line. Be
careful to only transfer the pigment onto the
origin line. Tip: Only prepare one strip with
sample at a time so that the sample does not dry
out.

and then remove the strip from the solvent.

Check on the strip and the solvent front every 5
to 10 minutes - if you let it run too long the dye
may run off the strip and become distorted. This
may take about 20 to 60 minutes.
Allow the chromatography strip to dry. Tip: An
easy way to do this is to tape the strip to the
overhang of a counter or table so that the strip is
dangling in the air.
After the strip has dried, measure the distance
(in centimeters) from the origin to the solvent
front and from the origin to the top of the
pigment band that should be visible, as shown in
Figure 4 below. Record the data in the data table
you made in your lab notebook.

Figure 2. Pigment is transferred to the strip.

Tape the strip to the pencil so that when you lay

the pencil across the top of the large-mouth glass
jar, as shown in Figure 3 below, the strip hangs
into the jar of solvent and the bottom edge of the
strip is just barely immersed in the solvent.
Note: The origin should not be immersed in the
solvent and be sure the strip hangs straight into
the solvent.

Figure 4. The pigment moves up the paper as the solvent

front advances. The Rf value is the ratio of the distance to the
top of the band, to the distance to the solvent front,
measured from the origin.

Note: If you see more than one pigment band on

the strip, record this band's color in your data
table in a new column titled "Color of the Second
Pigment Band." Also measure the distance from
the origin to the top of this pigment band and
record the data in your data table in a new
column, titled "Distance to the Top of the Second
Band (in cm)."
D. Variables
Try this experiment with other similar colors of
flowers, such as purple, pink, or orange flowers.

Figure 3. The setup for the paper chromatography jar.

Complete the setup as shown in Figure 3 and let

the solvent rise up the strip (by capillary action)
until the solvent front is about 2 cm from the top

You could try this science project but this time,

investigate colorful leaves on plants instead of
flowers. What pigments make the leaves so
colorful? Are these the same as the pigments in
similarly colored flowers?
E. Measurements

Calculate the Rf (Retention factor) value for each

pigment for each strip. Record the values in the
data table in your lab notebook.

distance
distance
Rf = top of pigment band (cm) solvent front(cm)

Retention factor is a fraction that is characteristic

for each pigment in a given set of experimental
conditions.
You can use Rf values to identify different
pigment molecules as long as the solvent, pH,
and type of paper remain the same.
Note: If you saw more than one pigment band on
a strip, calculate the Rf value for this band as
well and record it in your data table in a new
column titled "Retention Factor (Rf) of the
Second Band."
Compare the Rf values and colors of the pigment
bands for each different flower. To be the same
pigment, the pigment bands should have similar
Rf values and be a similar color.
F. Safety Precautions
If this experiment is being done by children, adult
supervision is necessary because of the use of
alcohol.
OBSERVATION

complex
mixture
or
solution.
In
paper
chromatography, a liquid like water or isopropyl
alcohol is the mobile phase (or solvent), and filter
paper is the stationary phase. A mixture is
dabbed onto a piece of filter paper near one end,
at a point called the origin. The same edge as the
mixture dab is immersed in the solvent, with the
origin above the level of the solvent. The paper
works like a wick, with the solvent moving up the
paper, due to capillary action. The pigment
molecules are then carried up the paper with the
moving solvent.
How does chromatography separate the different
molecules in a solution? The molecules ideally
move at different speeds as they travel through
the stationary phase. This is done by adjusting
the mobile and stationary phases so that they
interact with different properties of the solution's
molecules, such as their molecular size, electrical
charge, or other chemical properties, to
distinguish and separate them from each other.
In paper chromatography, different pigments can
be separated out from a solution based on the
solubility of the pigments. If a pigment is very
soluble in the solvent, it can easily be carried up
the paper with the solvent front, which is the
leading edge of the solvent, as it travels up the
paper. A pigment that is less soluble in the
solvent, or one that interacts with the filter paper
more than the solvent, will generally travel a
shorter distance. Because different pigment
molecules have different solubilities, they are
separated
from
each
other
on
the
chromatography paper.

The bright colors of flowers are primarily used to

attract pollinating insects. These bright colors are
produced by the pigments which are found in the
flowers.
There are two major classes of flower pigments:
carotenoids
and
flavonoids.
Carotenoids
include carotene pigments, which produce yellow,
orange, and red colors, and xanthophyll
pigments, which only produce yellow colors.
Carotene pigments are more commonly found in
vegetables, and, in fact, they are what make
carrots
look
orange.
Flavonoids
include
anthocyanin pigments, which produce red,
purple, magenta, and blue colors, and flavones
and flavonol pigments, which produce yellow
colors. Some flowers may even have chlorophyll,
a green pigment usually found in the leaves of
plants.
In this experiment, chromatography was the
technique used. Chromatography is a technique
that is used to separate out the components of a

Figure 5. Labelled diagram of the Chromatography apparatus

set-up.

CONCLUSION
As a result of the experiment, I am able to know
that a flower doesnt only have one pigment; it

can contain 2 or more. By the use of the

chromatography technique, I am able to identify
that pigments differ in solubility; therefore, they
are separated from each other on the
chromatography paper making the pigments
visible so you can be able to identify the pigment
and the color of the flower.
I also read that the light can affect the color of
the flowers because plants primarily produce
color by synthesizing pigments, which absorb
subsets of the visible spectrum, reflecting back
only what they dont absorb and causing the
tissue to be perceived as the reflected colors.
For example, chlorophyll absorbs light in both the
red and blue parts of the spectrum, reflecting
only the green light, and causes leaves to appear
green to us. Similarly, a flower that we perceive
as red contains pigments, which absorbs yellow,
green and blue light, leaving red light as the only
wavelength visible to us which is reflected.
EVALUATION
The results of the experiment gave answers as to
what gave the red flowers their color. There are a

lot of factors given and the chromatography

experiment proved to us that there are different
pigments in each flower.
REFERENCES
[1] Rowland, PhD, Teisha and Whyte, PhD, David.
Reveal the Red: Exploring the Chemistry of
Red Flower Pigments. 2013. [Retrieved from
<http://www.sciencebuddies.org/science-fairprojects/project_ideas/PlantBio_p035.shtml#
summary> on July 12, 2014]
[2] Science
Buddies.
Springtime
Science:
Exploring the Pigments in Flowers. 2013.
[Retrieved
from
<http://www.scientificamerican.com/article/b
ring-science-home-flower-pigment/> on July
12, 2014]
[3] Alkema, Joy and Seager, Spencer. The
Chemical Pigments of Plants. [Retrieved from
<http://courses.chem.psu.edu/chem35/plant
pig.pdf> on July 13, 2014]
[4] Brebbia, C.A., Collins, M.W., and Greated, C.
Colour in Art, Design and Nature. Great
Britain: WIT Press. 2011.