Separation of Photosynthetic Pigments using Paper Chromatography in San

Francisco (Codiaeum variegatum)

Encina, Erika G.
Fransisco, Sharmaine Joy C.
Garcia, Vincent Luis J.
Mapesos, Matthew Brandon G.
Pagayon, Daniel R.
Group 1 XI - STEM

July 12, 2016

__________________
A scientific paper submitted in partial fulfillment of the requirements in General
Chemistry laboratory under Mr. Fernando Desacola Rocapor-Posos Fabia Jr., 1 st sem.,
2016-2017.

ABSTRACT
Biological pigments found in plants whose main function are
to create food with the help of abiotic materials can be separated
from one another through the use of paper chromatography.
Chlorophyll, along with xanthophyll, plays a vital role in this
process. During photosynthesis, a process by which plants use
sunlight to manufacture foods from carbon dioxide and water, the
green pigment (chlorophyll) allows plant to absorb energy from the
sun while the yellow pigment (xanthophyll) plays an important
function as accessory pigments. Xanthophyll captures certain
wavelengths of sunlight not absorbed by chlorophylls. This results
to an increase in overall absorbance of the visible spectrum of
sunlight. Chromatography is a separation method that uses the
diversity of dissolved substances in terms of their degree of
attraction with a mobile phase and stationary phase. In this
experiment, the students separated the chloroplast pigments from
the leaves particularly of the San Francisco plant (Codiaeum
variegatum) by doing paper chromatography. With the use of filter
paper as the stationary phase and the mixture of alcohol and
acetone as the mobile phase, they successfully split the pigments
of the plant which are mainly chlorophyll and xanthophyll.

INTRODUCTION
Living organisms, especially plants, are known to produce substances called
biological pigments which are due to the organisms selective color absorption. In
autotrophs, the main function of these pigments is for food-making.
Chromatography is the separation of compounds by the differences in the way
those compounds interact with the separation environment. It is a resolution technique
that depends on the solubility differences of the components of a mixture in a given
solvent (General Inorganic Chemistry 1, pp. 61). It involves passing a mixture dissolved

in a "mobile phase" through a stationary phase, which separates the component of the
mixture to be measured from other molecules in the mixture and allows it to be isolated.
Separation can be effected by many different means including everything from high-tech
polymer-based resins to electric potentials to simple paper. These separations generally
take place in some sort of liquid for the mobile phase though some techniques involve
separation in gas. Chromatography is an area of science that many people have not
heard of but it is used in everything from pharmaceutical manufacturing to power
generation to genome sequencing. In most science applications chromatography is
used to separate components in a mixture either for analysis or for further use. An
example of the use of chromatography is the measurement of the amount of an active
ingredient in a pharmaceutical product. This separation might be done using revered
phase chromatography and UV detection in an organic solvent. The driving force behind
this separation is capillary action of the water wicking through the paper (General
Electronic Company, 2009).
Since chromatography splits the pigments of compounds, pigments within leaves
are also believed to be separable through the same process. In this experiment, the
students used filter paper as their chromatography media or stationary phase and
mixture of alcohol and acetone as their liquid or mobile phase to separate the different
pigments of San Francisco leaves. This test was designed to determine the pigments
that make up the leaves of San Francisco. Also, the pigments are presumed to go
upward and separate itself from the additional solvent added in. This was conducted at
the Science Laboratory of Sto. Rosario Sapang Palay College High School Department.

MATERIALS AND METHODS

The first step was to gather leaves of plants such as San Francisco (Codiaeum
variegatum), Bangka-bangkaan (Ephemerum bicolor) and other plants having
multicolored leaves. In this particular experiment, the students chose San Francisco
with yellow and green color. For the filter paper, the circular piece was cut into a thinner
strip which is about half-inch in width. The students measured one inch from the end
part and marked it with a penciled line. The chosen leaves were cut into small pieces
using scissors. The pieces of leaves were later on transferred to a mortar and were
pounded with a pestle. Gradually, alcohol and acetone was poured in. The pounding
was continuous until the pigment started to come out and incorporated itself with the
liquid mixture. In order to get more of the juice within the leaves, they squeezed the
leaves and removed it from the mortar. The liquid mixture was put in a petri dish. After
that, they created an improvised dropper by just using the end part of the remaining
leaves. The tip was soaked in the extract. A droplet sticked at the end and it was
dropped in the center of the same spot where the line was drawn. They did it for
multiple times enough to ensure that the pigment was adequate to see the pigments
better after it dries up. Through air-drying, they waited until the paper dried up.
Meanwhile, they prepared the beaker where the filter paper will be soaked into. To do
this, they searched out for a beaker with a wide mouth for the paper to fit in. Next, the
paper was taped to the pencil to create a hanger-like set-up at the top. The beaker was
then filled with alcohol. The students put alcohol with an amount not reaching the
pigment to avoid dissolving it. For about an hour, they observed the specimen.

RESULTS AND DISCUSSION

The experiment showed the separation of the green,

brown and the yellow pigment of the leaves of San Francisco
(Codiaeum variegatum) by the use of solvents acetone
(C3H6O) and alcohol (C2H6O). The pigments chlorophyll a
(green pigment), chlorophyll b (yellow-green pigment),
xanthophyll (yellow pigment), phaeophytin b (yellow-brown
pigment), appeared in the filtration paper.
For about an hour, they observed as the pigment slowly went up and as the
green and yellow pigment separated from one another with the help of capillary action.
This action pulled the solutes (pigment) up through the paper and separated them.
Chromatography works because different substances in a mixture have different
degrees of solubility. Solubility refers to how much of a particular substance can
dissolve in a particular solvent. The chromatography paper indicated that the green and
yellow leaf of Codiaeum variegatum is composed of green, yellow-green, yellow, and
yellow brown pigments.

SUMMARY AND CONCLUSION

Chromatography is the process of separating the pigments in relation to the
differences

in

the

ways

the

compounds

interact

with

one

another. Paper

chromatography is one simple way of color separation by just using everyday materials
like alcohol, acetone and a paper. This experiment gives insights to what pigments
multi-colored leaves are composed of. The initial data from chromatography can lead to
many other experiments and connections made between photosynthesis and leaf
pigmentation.
LITERATURE CITED

Engr. Dimayacyac, M.D., Engr. D. Grace, Engr. E.E. Delena, Engr. R. Gasendo,
Engr. R. Pimentel, Prof. A. Bayquean, Prof. C. Centeno. 2015. General Inorganic
Chemistry 1. 1st ed. Espana, Manila:,University of Santo Tomas. pp. 61
General Electric Company (2009) Paper Chromatography With Standards [Online]
Available:
(http://www.ge.com/press/scienceworkshop/docs/pdf/Paper_Chromatography_wit
h_Standards.pdf) [2016, August 4]. (Date accessed)