COLUMN CHROMATOGRAPHY OF PLANT PIGMENTS

De Jesus, Joshua L., Bonifacio, Raymond B., Cabatbat, Jay Carlo, Cayco, Emil
Florencio G., Cortez, Ma. France
Abstract: The general objective of the experiment was to separate and identify plant
pigments present in siling labuyo. The method used in the experiment was column
chromatography. Through this method, the orange and yellow pigments were obtained.
The difference in the equilibrium constants for the distribution of each component
between the two phases resulted in separation. The molecules of the component which
was held less strongly by the stationary phase which moved over at a higher rate than the
component which was held more strongly. As a result, components migrated into separate
regions of the stationary.
I. INTRODUCTION
In column chromatography, the stationary phase, a solid adsorbent, is placed in a
vertical glass (usually) column and the mobile phase, a liquid, is added to the top and
flows down through the column (by either gravity or external pressure). Column
chromatography is generally used as a purification technique: it isolates desired
compounds from a mixture.
The mixture to be analyzed by column chromatrography is applied to the top of
the column. The liquid solvent (the eluent) is passed through the column by gravity or by
the application of air pressure. An equilibrium is established between the solute adsorbed
on the adsorbent and the eluting solvent flowing down through the column. Because the
different components in the mixture have different interactions with the stationary and
mobile phases, they will be carried along with the mobile phase to varying degrees and a
separation will be achieved. The individual components, or elutants, are collected as the
solvent drips from the bottom of the column.
Column chromatography is separated into two categories, depending on how the
solvent flows down the column. If the solvent is allowed to flow down the column by
gravity, or percolation, it is called gravity column chromatography. If the solvent is
forced down the column by positive air pressure, it is called flash chromatography, a
"state of the art" method currently used in organic chemistry research laboratories The
term "flash chromatography" was coined by Professor W. Clark Still because it can be
done in a flash."

II. METHODOLOGY
1. Extraction of Plant Pigment
Extraction was done by deseeding three siling labuyo. After deseeding, it was
grinded using a mortar and pestle. Then, 3 ml. Of DCM (dichloromethane) was added.
The mixture was then decanted and transferred into a vial.
2. Column Packing (micro scale)
In this step, a small piece of cotton was plugged inside the dropper. The size of
the cotton was just enough so that it can act as a stopcock. After plugging in the cotton,
the dropper was then secured in an iron stand using a burette clamp. After which, silica
gel was added inside, just an adequate amount to fill the dropper up to the indented part.
3. Column Loading
The sample inside the vial was then quickly placed inside the dropper. The
transferring of the sample to the dropper was done quickly to prevent evaporation.
4. Column Elution
After the sample was absorbed, the first eluting solvents was then used to drain
the column. The eluting solvents used were hexane, DCM: hexane, DCM, and DCM:
methanol. Two ml. of each was continually added respectively, not allowing the dropper
to run dry, until a color band descended. The pigments that eluted out of the column were
collected in separate vials depending on the color. The number of drops were also
recorded.

Fig. 1 Plugging the cotton

Fig. 2 Adding silica gel

Fig. 3 Securing dropper in burette clamp

Fig. 4 Collecting the pigments

III. RESULTS AND DISCUSSION

Three pigments were collected after the experiment, the yellow, the orange and
the red. Yellow came out first. Colorless drops followed before the orange pigment
descended. After which, the red pigment came out.

Color of Eluate

Number of Drops

Pigment Name

YELLOW

124

CUCUBITINE

ORANGE

38

CAROTENE

RED

47

CAPSANTHIN,
CAPSORBIN

The color of siling labuyo in ripe state is mainly from carotenoid pigments which
range from bright red (capsanthin, capsorbin and more) to orange (carotene) to yellow
(cucubitine).
IV. CONCLUSION
The experiment shows that it is best to use column chromatography in extracting
plant pigments. Color of pigments vary. The descending of pigments is based on the
adsorption of solute to the adsorbent and the eluting solvents flowing through the column.
V. REFERENCES
www.wikipidia.org
www.encyclopedia.com
Organic Chemistry by Wilhelm Von Snitzki, pp. 66-84

Chemistry For the Ages by Sean Pierre de Lemar, pp. 345-350