CHROMATOGRAPHY

Myrabelle B. Lopez
CHEM 31.1- BC

ABSTRACT

Plant pigments are capable of absorbing visible light, which can be used in order to
harvest energy for photochemical reactions. There are a variety of pigments present in plants
such as carotenoids, flavonoids. anthocyanin and chlorophyll. For this experiment, the Aloe
vera leaf was subjected to paper chromatography which is a method used by separating
dissolved chemical substances by taking advantage of their different rates of migration
across sheets of paper. The chromatogram revealed that the only pigment extracted was
chlorophyll which is essential in the photosynthetic mechanism of the plant and is responsible
for its green appearance likewise, the solvent mixture of 9:1:1 (v/v) of hexane: ethanol :
acetone proved to be a better mobile phase compared to the 9:1 (v/v) of ethanol and acetone.
I. INTRODUCTION

The science of organic chemistry contains a wide array of experiments that will allow
us in analyzing different organic compounds which are part of our everyday lives.
Chromatography is one it. It is a technique for the separation of a mixture by dissolving it in a
fluid called the mobile phase, which carries it through a structure holding another material
called the stationary phase. The various constituents of the mixture travel at different speeds,
causing them to separate (Principles of chromatography, n.d.). There are many types of
chromatography namely: column, planar, paper and TLC but for this experiment, paper
chromatography will be utilized.

According to Britannica, T. E. (2018) it is a chromatographic technique for separating

dissolved chemical substances by taking advantage of their different rates of migration across
sheets of paper. It involves placing a small dot or line of sample solution onto a strip of
chromatography paper which is placed in a container with a shallow layer of solvent and
sealed. As the solvent rises through the paper, it meets the sample mixture, which starts to
travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and
the compounds within the mixture travel farther if they are non-polar. More polar substances
bond with the cellulose paper more quickly, and therefore do not travel as far. Paper
chromatography is an example of liquid-liquid partition chromatography which helps in the
rapid analysis of the components of reaction mixtures and as a tentative way of identification.

Plant pigments can be subjected to paper chromatography. A "pigment" is simply a

molecule that absorbs and reflects light. Different pigments appear different colors because
they have differing abilities to absorb and reflect various colors of light. Some essential
pigments are chlorophyll which 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;
carotenoids are usually red, orange, or yellow pigments and they cannot transfer sunlight
energy directly to the photosynthetic pathway, but must pass their absorbed energy to
chlorophyll and lastly is the phycobilis which is a water-soluble pigment found in the
cytoplasm, or in the stroma of the chloroplast which occur only
in Cyanobacteriaand Rhodophyta. (Photosynthetic pigments., n.d.).
 By using paper chromatography in the separation of plant pigments, this experiment is
designed to learn the techniques of paper chromatography and apply chromatographic
methods in the separation of the components of a mixture.

II. RESULTS

The following tables shows the data gathered through the paper chromatography of
plant pigments.

Table 1. Data on the plant sample

Scientific Name Aloe vera

Common Name Aloe vera
Zone 2 Pastileros Subdivision, Brgy.
Place and Date of Collection Guindapunan Palo, Leyte collected on
February 21, 2019
It has fleshy leaves which are colored green
Description of Sample
with spines surrounding it.
It has viscous texture which is light green in
Description of Acetone Extract
color.

Table 2. Data on the analysis of plant pigments by paper chromatography

Solvent system 9:1:1 9:1

Sketch of
chromatogram
III. DISCUSSIONS

Table 1 reflects the data on the plant sample- Aloe vera. Due to having fleshy leaves,
it was easy to extract its leaf pigments which were colored light green and has a gelatinous
texture and appearance.

In Table 2, the chromatogram reveals that the extracted pigment was chlorophyll due
to its light green appearance. Chlorophyll is a pigment which is critical for the light reactions
of photosynthesis. It was separated by using two sets of 5 mL solvents : 9:1:1 of hexane:
ethanol and acetone then 9:1 of hexane and acetone. Both solvents contain acetone since it is
an amphipathic substance that has both polar and non-polar ends which allows it to dissolve
non-polar substances and has the ability to travel up the paper hence creating a greater
resolution between pigments on the paper (Function Of Acetone In Chromatography
Experiment., n.d.). However the first solvent system which is 9:1:1 (v/v) of hexane:
ethanol:acetone is more effective since it contains ethanol, a polar molecule with OH bond
causing this solvent to be strongly attracted to the paper allowing more pigments to be carried
as it rises. This reaction is proven true from the observed distance travelled by the spot from
the origin which is 4 cm. The second solvent system only contains hexane, a non-polar
compound and acetone which is not strongly attracted to the paper due to its lack of hydrogen
bonding which caused the pigments to only travel 1.5 cm from its origin .

IV. CONCLUSIONS

After conducting the liquid-liquid extraction, the following were the significant
results and implications gathered.

 Paper chromatography is an effective method of separating and observing the

various colors of plant pigments. The pigments dissolved in the solvent and
migrated upward.
 The only observed pigment was chlorophyll.
 The solvent mixture of 9:1:1 (v/v) of hexane: ethanol : acetone proved to be a better
mobile phase compared to the 9:1 (v/v) of ethanol and acetone.

ANSWERS TO QUESTIONS
1. What would be the effect of the following errors in chromatographic work?

a. The solvent level in the developing chamber is higher than the spotted sample?

The solvent will dissolve the spotted compounds into the solvent instead of
allowing the compounds to move up the plate by capillary action hence, no spots will
appear after development. (Judge, M., 2019)

b. Too much sample is applied to the paper

Applying too much sample may result in large, diffuse bands of chemical
moving up the plate, making it difficult to accurately measure the distance the
chemical has been transported. (Judge, M., 2019)

c. The paper is allowed to remain in the chamber after the solvent front has reached
the top of the plate.

The retention factor of the solvent cannot be calculated since the distance of
the solvent front from the origin was unknown. (Judge, M., 2019)

2. Why is it necessary to cover the developing chamber tightly during the development of a
chromatogram?

It should be tightly covered during the development of the chromatogram to

ensure that the solvent where it is subjected will not evaporate. (Litteulbaek., n.d.)

3. Can TLC or paper chromatography be used to separate and identify very volatile
substances? Explain your answer.

It cannot be used to separate and identify very volatile substances since these
volatile substances will only be removed during the evaporation of the developing
solvent. (Litteulbaek., n.d.)

4. In Part C, why were you required to handle the chromatographic paper only at its
corners?

There might be a possible imprint due to the reaction between the chemicals
from the hands and the sample and solvent. (Litteulbaek., n.d.)

REFERENCES
Britannica, T. E. (2018, June 01). Paper chromatography. Retrieved March 4, 2019, from
https://www.britannica.com/science/paper-chromatography

Function of Acetone In Chromatography Experiment. (n.d.). Retrieved March 4, 2019, from

https://www.enotes.com/homework-help/why-acetone-good-solvent-use-
when-testing-pigments-323891

Judge, M. (2019, March 02). Factors That Affect RF Values in Thin Layer Chromatography.
Retrieved March 10, 2019, from https://sciencing.com/factors-values-thin-
layer-chromatography-8561359.html

Litteulbaek. (n.d.). C the paper is allowed to remain in the chamber after. Retrieved March
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Photosynthetic pigments. (n.d.). Retrieved March 3, 2019, from

https://ucmp.berkeley.edu/glossary/gloss3/pigments.html

Principles of chromatography. (n.d.). Retrieved March 3, 2019, from

https://www.khanacademy.org/test-prep/mcat/chemical-
processes/separations-purifications/a/principles-of-chromatography