Objective

Analyze the pigments in red flower petals and determine if different

red flowers use the same or different pigments.

Introduction
Plants contain many complex chemicals that have different functions.
Some of these chemicals are used in the normal metabolism of the
plant, such as those involved in photosynthesis.

Some are involved in the plant's defense against insects, bacteria, and
other pathogens. And some, such as the pigments in a flower, are
involved in attracting the attention of possible pollinators, such as
honeybees, butterflies, and hummingbirds.

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.

➢ 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.

➢ The goal of this plant biology science fair project is to analyze

the pigments found in flowers using paper chromatography.
Materials and Equipment
✓ Candy Chromatography Science Kit (1). You will need these items
from the kit
✓
✓ Chromatography paper strips; 9. The kit comes with 20 strips;

✓ 100 mL beaker

✓ 90% isopropyl alcohol

✓ Mini binder clips (2)

✓ Wooden splint

You will also need to gather these items

✓ 100 mL graduated cylinder or measuring cup.

✓ Clean jar, drinking glass, or mug
✓ Scissors
✓ Ruler, metric
✓ Pencils (2)
✓ Distilled water (at least 60 mL). Distilled water is preferable, but
tap water is also suitable
✓ Flower petals, red; you will need at least 2 flower petals from at
least 3 different plants, which you can get from your own garden or
from a florist or plant nursery. Tip: Larger petals, such as those
from roses and tulips, work better than smaller petals.
✓ Piece of scratch paper
✓ Coin
✓ Timer or clock
✓ Lab notebook
 Experimental Procedure
To make sure you can compare your results, as many of your materials
as possible should remain constant. This means that the temperature,
type of water used, size of paper strips, where the ink is placed onto
the paper, etc., should remain the same throughout the experiment.

Cutting and Marking the Paper Strips

1. Cut each chromatography paper in half (length-wise) to make

approximately 2 centimeters (cm) wide by 7.5 cm long strips. You will
need at least 9 chromatography strips.

2. In your lab 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. This is so that you can identify which flower was used
with which strip later.
a. If you are investigating more than three different types of flowers,
similarly continue to number the test strips.

3. Draw a pencil line 1 cm from the edge of each strip of paper

a. This will be the origin line or baseline.
b. You will spot the flower petal pigment for each strip right on this line.
Performing Paper Chromatography on the Paper Strip

• In your lab 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 the jar, dilute the 90% isopropyl alcohol by mixing

75 milliliters (mL) of water with 30 mL of isopropyl alcohol. This
water and isopropyl alcohol mixture is your solvent.

• Pour a small amount of the solvent into your 100 mL beaker, about
1–2cm cm deep.

• Next, transfer the pigments from one type of flower onto a strip
of chromatography paper.

• Clip the prepared chromatography strip to a wooden splint. Rest

the splint on top of the beaker so that the strip hangs straight
into the beaker. Note: The origin should not be immersed in the
solvent.

• If necessary, add more of the solvent. The goal is to have the end
of the chromatography strip just touching the surface of the
solvent solution,.
• Let the solvent rise up the strip (by capillary action) until the
solvent front is about 2 cm from the top 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 pigments may run off the strip and
become distorted.

• Use a pencil to mark the solvent front.

• 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.

• Repeat steps 4–10 two more times for the same type of flower.

• Repeat steps 4–10 two more times, each time with a different
type of flower, so that you have run at least three
chromatography paper strips for each of the three different
flower types you want to investigate.
Observations and results

• Carotene pigments (which are carotenoids) produce yellow, orange

and red colors whereas anthocyanin pigments (which are flavonoids)
produce red, purple, magenta and blue colors. Most red flowers use
anthocyanin pigments to produce their red coloring (although some
use carotenoids). On the paper strips, the anthocyanin pigments may
have appeared as a purplish-reddish band.

• In paper chromatography the pigments move up the paper with the

liquid and are separated based on the solubility of the pigments. So,
if a pigment is very soluble in the diluted isopropyl rubbing alcohol,
it should be easily carried far up the paper strip whereas a less
soluble pigment will generally travel a shorter distance. Because
different pigments often have different solubilities, they can be
separated from one another on the paper strip.
 Experiment with Paper Chromatography
• Chromatography is a technique used to separate a mixture or
solution into its individual components. There are several
different types of chromatography, including thin-layer, column,
and paper chromatography.

• Paper chromatography helps students discover the color

molecules used to make the color our eyes perceive in a candy
coating, for example, or a marker or bottle of ink.

• chromatography, three different brands of black water-based

markers may all show different color molecules in the ink.
Similarly, candies of a specific color may all look the same, but
paper chromatography may reveal that their composition is very
different.

• In a paper chromatography experiment, a small amount of a

sample solution is placed on a strip of chromatography paper. The
chromatography paper is then suspended in a solvent.

• As the solvent moves up the paper, the individual components of

the sample solution separate out in bands of individual color.
ADVANTAGE OF RED FLOWER PIGMENT

• Carotenoids (specifically lycopene), anthocyanins, and betacyanins are

natural red pigments found in fruits and vegetables.
• They possess antioxidant properties beneficial in the prevention of
cancer and cardiovascular diseases. Recently, natural colouring agents
have attracted increasing attention from the food industry as a
substitute to the artificial colouring compounds, which have been
discovered to cause negative impact to human’s health upon
consumption.
• There is a growing interest in the natural red pigments in food science,
due to their preventive effects on chronic diseases. Stability of the
natural pigments should be concerned, as it might affect the
saturation of the colour.