EXERCISE 4

PIGMENT ANALYSIS VIA PAPER CHROMATOGRAPHY

Introduction

Photosynthesis begins when light is absorbed by pigments in the plant cell. One technique
for separating and identifying these pigments is paper chromatography. In paper chromatography,
solvent moves up the paper carrying with it dissolved substances - in this case, plant pigments. A
pigment is a molecule that absorbs light. Chromatography is a method of separating and isolating
molecules based on their level of hydrophobic or hydrophilic properties. In paper chromatography,
we create a “molecular race track” in which molecules move through a piece of filter paper, carried
along by a wave of liquid solvent. Those pigment molecules that have the highest solubility in the
liquid solvent used will be “carried along” through the paper the fastest. Those pigments that are
least soluble in the solvent will move more slowly or not at all.

Objective
To demonstrate the separation of plant pigments from plant leaves

Note: For this activity, you have the following options:

1. If you have the materials, you can perform the activity and answer the questions based on
your results.
2. If the materials are not available in your home, you can watch the video provided on this
activity and then answer the questions based on your observation on the results.

Materials

 Leaves  Chromatography or filter

 Beaker or any glass container paper (you can use coffee filters
or paper towel/tissue)
 Isopropyl (rubbing) alcohol
 Pens or Pencils
 Plastic wrap

Procedure

1. Grind the leaves and place them in a beaker or glass, then add just enough rubbing alcohol
to cover them. Cover the beaker with plastic wrap to keep the alcohol from evaporating.

2. Put the beaker in a dish of hot tap water for about 30 minutes, until the alcohol turns green
as the pigments from the leaves are absorbed into it.
 3. Cut a strip of filter paper (Tissue paper) about a half inch wide and tape it to a pencil.
Suspend the pencil across the beaker and let the strip just barely touch the alcohol and
pigment mixture. (Make sure your paper does not touch the glass side or other papers)

4. Allow the filter-paper (Tissue paper) strips to dry. Some possible colors and the pigments
they represent are:

faint yellow / orange - carotenes olive green - chlorophyll b

yellow – xanthophyll red - anthocyanin
blue green - chlorophyll a

5. Record your result.

(For option 2, please watch this video https://www.youtube.com/watch?v=qH-AJDqsSII on

YouTube and answer the questions based from the results they obtained. The procedure in this
video is almost similar to the procedure in Exercise 4.)
 Exercise No. 4
PIGMENT ANALYSIS VIA PAPER CHROMATOGRAPHY

I. Questions:

1. How many pigments can you visualize?

I can visualize three pigments which are green, faint yellow, and orange.

2. What plant pigment molecules might account for the different color pigments you observed?

Chlorophyll is the plant pigment molecule responsible for green pigment, while carotenoids
are responsible for both faint yellow and orange color.

3. Do the pigments absorb all wavelengths equally or do they exhibit differential absorption?

The pigments exhibit different absorptions.

4. Why are leaves green even though other pigments are present?

Green pigment is noticeable because green light is reflected rather than absorbed by the
leaves. On the other hand, light of other colors, such as blue and red, is absorbed.

II. Conclusion
The goal of this experiment is to utilize paper chromatography to separate the different
compounds within the leaves. This experiment revealed that chloroplast contain a variety of pigments
that are all involved in the collection of energy from sunlight. The pigments and their absorption were
represented on the filter paper as a range of color. As stated in the video, leaf pigments interact
differently on the coffee filter based on the chemical structure of the leaf. Some pigments will cling to
the paper tightly, while others will flow more freely with the liquid.
 DOCUMENTATION

Figure 1. Materials

Figure 2. Pouring Isopropyl Alcohol on the Glasses

Figure 3. Hot Water Bath to Speed up The Process

Figure 4. Placing the Coffee Filter Strips into each Glass

Figure 5. Separation between Yellow Carotenoids and

Green Chlorophyll