Separating Pigments of Chile Pepper Using Column Chromatography and Thin

Layer Chromatography

Freida Coning, Tanya Cruz, Patricia dela Pasion, Joyce De leon, Norina Dimalibot

Group 4, 2F Pharmacy Organic Chemistry Laboratory

Abstract

Chromatography is a method for analyzing complex mixtures by separating them into the chemicals from
which they are made. In this experiment, pigments of the Chile Pepper/Cayenne (siling labuyo in Filipino)
with the scientific name Capsicum frutescens, were extracted with the use of DCM-hexane(with mole ratio
1:1). Extract was introduced into the column and eluate was collected; this process is the column
chromatography method. The purity of the components was determined by using thin layer chromatography.
Ultraviolet lamp was used to visualize the developed TLC plate(from the thin layer chromatography) and the
Retention Factor was measured for each pigment.

Introduction plate and the moving solvent, thus

separating the components of the mixture so
Chromatography is defined as a process that the components are separated into
used to separate mixtures of substances into separate "spots" appearing from the bottom
their components. They all have to the top of the plate.In gas
a stationary phase (a solid, or a liquid chromatography, the stationary phase is a
supported on a solid) and a mobile high-boiling liquid which is packed into a
phase (a liquid or a gas). long, narrow glass or metal column. The
mixture to be analyzed is loaded by syringe
There are several types of into the beginning of this column. The mobile
chromatography. In column chromatography, phase is an inert gas which continuously
the stationary phase is a powdered flows through the column. The components
adsorbent which is placed in a vertical glass of the mixture distribute between the
column. The mixture to be analyzed is loaded stationary high-boiling liquid (these
on top of this column. The mobile phase is a components are either condensed or
solvent poured on top of the loaded column. absorbed on the high-boiling liquid) and
The solvent flows down the column, causing mobile gas (vapor) phase moving through
the components of the mixture to distribute the column. The gaseous mixture flows
between the powdered adsorbent and the through a detector at the end of the column
solvent, thus (hopefully) separating the and if it has been successfully separated, the
components of the mixture so that as the components show as different 'blips' or peaks
solvent flows out of the bottom of the on a recorder. In chromatography,
column, some components elute with early the retardation factor (also known
collections and other components elute with as retention factor) describes the ratio of
late fractions. In thin layer chromatorgraphy, time spent in the stationary phase relative to
the stationary phase is a powdered adorbent time spent in the mobile phase.
which is fixed to a aluminum, glass, or plastic
plate. The mixture to be analyzed is loaded In the experiment, the group used column
near the bottom of the plate. The plate is and thin layer chromatography.
placed in a reservoir of solvent so that only
the bottom of the plate is submerged. This
solvent is the mobile phase; it moves up the
plate causing the components of the mixture
to distribute between the adsorbent on the
 liquid. This liquid, or the eluent, is the mobile
phase, and it slowly rises up the TLC plate by
capillary action. The Retention factor is
computed using this formula:

Figure 1. Column Chromatography set-up.

In column chromatography, the mixture to

be analyzed is applied to the top of the
column. The liquid solvent (the eluent) is
passed through the column by gravity or by Figure 2. TLC set-up.
the application of air pressure. An
equilibrium is established between the solute The objective of the group’s experiment is
adsorbed on the adsorbent and the eluting to separate the colored components of the
solvent flowing down through the column. Chile pepper using column chromatography,
Because the different components in the to determine the purity of the pigments using
mixture have different interactions with the thin layer chromatography and to measure
stationary and mobile phases, they will be the retardation or retention factor of each
carried along with the mobile phase to component.
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. The
Experimental
retardation factor, R, is the fraction of the
sample in the mobile phase at equilibrium. It
is expressed as The Chile peppers were triturated using the
mortar and pestle until delapitated, and then
eventually the group added 7mL DCM-
hexane(1:1) was poured. The mixture is
triturated 2-3 times and then the extract
Thin Layer Chromatography(TLC) is a from it is collected using a vial, covered and
simple, quick, and inexpensive procedure was set aside first, to prepare the column
that gives the chemist a quick answer as to chromatography set up.
how many components are in a mixture. TLC
is also used to support the identity of a Using the Pasteur pipette with the cotton in
compound in a mixture when the Rf of a it for bed support, the group filled it with
compound is compared with the Rf of a silica up to the indented portion of the glass.
known compound (preferrably both run on The pipette is clamped to the iron stand and
the same TLC plate). the test tubes are prepared as receivers.
The group also prepared the solvents. One
A TLC plate is a sheet of glass, metal, or test tube contains 0.5mL of DCM-
plastic which is coated with a thin layer of a hexane(1:1), another for another 0.5mL
solid adsorbent (usually silica or alumina). A DCM only, and another test tube for 0.5ml
small amount of the mixture to be analyzed DCM-methanol(1:1)
is spotted near the bottom of this plate. The
TLC plate is then placed in a shallow pool of One milliliter of the extract was introduced
a solvent in a developing chamber so that to the pipette. As the liquid goes down, it is
only the very bottom of the plate is in the
eluated by the first solvent, the 2mL DCM- For column chromatography, as we poured
hexane, followed by the 2mL DCM and lastly the extract and then the eluents(solvents) to
the 2mL DCM-methanol. The group changes the pipette, four eluates/colored pigments
the receiver each time the color varies. The were yielded. These were the colors yellow,
number of drops per pigment is also noted. dark orange, orange and light pink
After collecting all the eluates, Thin Layer respectively.
Chromatography was performed.

The eluates were applied on the TLC

plate(5cm x 8cm) by equidistantly spotting
each pigment ten times. Each spot was
allowed to dry first before applying the
succeeding spots. It was ensured that the
spots are made small so that when the plate Figure 4(left) and 5(right). Column Chromatography set
develops, the colors would not disarray. up and the eluates collected.

The developing chamber was prepared by Table 1. Column Chromatography results

placing the approximate amount of DCM- COLOR OF VOLUME (number

hexane to a beaker. The inner wall was lined COMPONENT of drops)
by filter paper to allow the TLC plate to 1 Yellow 100 drops
stand, and then covered with watch glass for 2 Dark Orange 29 drops
few minutes for equilibration. 3 Orange 33 drops
4 Light Pink 23 drops
After equilibrating, the developing plate
was carefully introduced to the developing
The volume of the yellow pigment was 100
chamber. The solvent system was allowed to
drops, the dark orange was 29 drops, orange
rise up until it reaches just one centimeter
was 33 drops and light pink was 10 drops.
from the upper end. The developing plate
was then removed from the chamber
carefully, and the group marked the solvent
fronts of each pigment and then air-dried the
plate. The components were visualized and
checked by the UV lamp. The retention
factors were measured after.

Results and Discussion

Chile Pepper/Cayennes(siling labuyo in

Filipino) was the specimen used for the
experiment. Dichloromethane-hexane is used
as the solvent system. The extract collected
from the six cayennes was three milliliters.

Figure 6. Thin Layer Chromatography product.

With reference to Figure 6, after collecting

the eluates, the group started the Thin Layer
Chromatography by spotting each pigment to
the TLC plate, putting the extract first on the
first spot, second is the yellow pigment, third
is the dark orange and last is the light pink.
Then the group prepared the TLC chamber
Figure 3. The extract from Chile pepper. and equilibrated it first before placing the
TLC plate. It is important to equilibrate the
set up first because this ensures complete
distribution of the solvent in the chamber.
References
As the TLC plate was introduced, the group
observed as the components went up the Robards K., Haddad, P. R., Jackson, P. E.,
plate with the solvent. (1994). Principles and Practice of Modern
Chromatographic Methods. San Diego, CA:
Table 2. Thin Layer Chromatography Results
Academic Press Inc.
COLOR OF Distance of RETENTION
COMPONENT the FACTOR Pavia, D. L., Lampman, G. M., Kriz, G. S., &
component Engel, R. G. (1999). Organic Laboratory
from the echniques: A Microscale Approach(3rd Ed.).
origin in cm Hardcourt College Publisher.
Yellow 6cm 0.92
Dark Orange 5.3cm 0.81 Fedessender, J. S., Fedessender R. J., & Feist
Orange 1.2cm 0.18 P. (2001) Organic Laboratory Techniques.
Light Pink 0 0 Canada:Brooks.

After the TLC plate was dried and viewed Pastro, D. J., John, C. R., & Miller, M. S.
from the UV lamp to mark the pigments, the (1998). Experiment and Technology in
group immediately measured the distance Organic Chemistry. New Jersey: Prentice
travelled by the components from the origin Hall.
to the solvent front. The distance travelled
by the extract was 4cm therefore having a Williams, T. I. (1997). An Introduction to
0.61 Rf value. Chromatography. New York: Chemical
Publishing Co., Inc.
Also based from table 2, the Retardation
factors were computed by dividing the Retrieved August 11, 2010
distance travelled by the solute or the http://orgchem.colorado.edu/hndbksupport/
compound from the distance travelled by the TLC/TLC.html
solvent. The solvent travelled up to 6.5 cm.
Therefore, for the Yellow pigment, if you Retrieved August 11, 2010
divide 6 by 6.5, you will get the retention http://www.chemguide.co.uk/analysis/chrom
factor of 0.92. Same formula was used for atography/paper.html
the remaining pigments that’s why the group
got 0.81 retention factor for the dark orange
pigment, 0.18 for the orange and zero for
the light pink pigment. The light pink
pigment failed to develop a retention factor
because the group didn’t have enough of the
eluate for the ten rounds of spotting.

The group learned that one of the

processes involved in the experiment was
Elution, which is the method of extracting
one material from another, usually by the
means of a solvent. The solvent is called the
eluent,and the pigments are called the
eluates.