Separating Pigments of Chile Pepper Using Column Chromatography and Thin Layer Chromatography

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

and thin layer chromatography.
compound is compared with the Rf of a

known compound (preferrably both run on
the same TLC plate).
A TLC plate is a sheet of glass, metal, or
plastic which is coated with a thin layer of a
solid adsorbent (usually silica or alumina). A
small amount of the mixture to be analyzed
is spotted near the bottom of this plate. The
TLC plate is then placed in a shallow pool of
a solvent in a developing chamber so that
only the very bottom of the plate is in 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
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. The retardation
factor, R, is the fraction of the sample in the
mobile phase at equilibrium. It is expressed
as
Figure 2. TLC set-up.
The objective of the groups experiment is

to separate the colored components of the
Chile pepper using column chromatography,
to determine the purity of the pigments using
thin layer chromatography and to measure
the retardation or retention factor of each
component.
Experimental
Thin Layer Chromatography(TLC) is a
simple, quick, and inexpensive procedure
that gives the chemist a quick answer as to
how many components are in a mixture. TLC
is also used to support the identity of a
compound in a mixture when the Rf of a
The Chile peppers were triturated using the

mortar and pestle until delapitated, and then
eventually the group added 7mL DCMhexane(1:1) was poured. The mixture is
triturated 2-3 times and then the extract
from it is collected using a vial, covered and

was set aside first, to prepare the column
chromatography set up.
plate. The components were visualized and

checked by the UV lamp. The retention
factors were measured after.
Using the Pasteur pipette with the cotton in

it for bed support, the group filled it with
silica up to the indented portion of the glass.
The pipette is clamped to the iron stand and
the test tubes are prepared as receivers.
The group also prepared the solvents. One
test tube contains 0.5mL of DCMhexane(1:1), another for another 0.5mL
DCM only, and another test tube for 0.5ml
DCM-methanol(1:1)
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.
One milliliter of the extract was introduced

to the pipette. As the liquid goes down, it is
eluated by the first solvent, the 2mL DCMhexane, followed by the 2mL DCM and lastly
the 2mL DCM-methanol. The group changes
the receiver each time the color varies. The
number of drops per pigment is also noted.
After collecting all the eluates, Thin Layer
Chromatography was performed.
Figure 3. The extract from Chile pepper.
For column chromatography, as we poured

the extract and then the eluents(solvents) to
the pipette, four eluates/colored pigments
were yielded. These were the colors yellow,
dark orange, orange and light pink
respectively.
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
develops, the colors would not disarray.
The developing chamber was prepared by
placing the approximate amount of DCMhexane to a beaker. The inner wall was lined
by filter paper to allow the TLC plate to
stand, and then covered with watch glass for
few minutes for equilibration.
After equilibrating, the developing plate
was carefully introduced to the developing
chamber. The solvent system was allowed to
rise up until it reaches just one centimeter
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
Figure 4(left) and 5(right). Column Chromatography set

up and the eluates collected.
1
2
3
4
COLOR OF
COMPONENT
Yellow
Dark Orange
Orange
Light Pink
VOLUME (number
of drops)
100 drops
29 drops
33 drops
23 drops
Table 1. Column Chromatography results
The volume of the yellow pigment was 100

drops, the dark orange was 29 drops, orange
was 33 drops and light pink was 10 drops.
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
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.
As the TLC plate was introduced, the group
observed as the components went up the
plate with the solvent.
COLOR OF
COMPONENT
Yellow
Dark Orange
Orange
Light Pink
Distance of
the
component
from the
origin in cm
6cm
5.3cm
1.2cm
0
RETENTION
FACTOR
0.92
0.81
0.18
0
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 didnt 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.
Table 2. Thin Layer Chromatography Results
After the TLC plate was dried and viewed

from the UV lamp to mark the pigments, the
group immediately measured the distance
travelled by the components from the origin
to the solvent front. The distance travelled by
the extract was 4cm therefore having a 0.61
Rf value.
References
Also based from table 2, the Retardation

factors were computed by dividing the
distance travelled by the solute or the
compound from the distance travelled by the
solvent. The solvent travelled up to 6.5 cm.
Therefore, for the Yellow pigment, if you
divide 6 by 6.5, you will get the retention
factor of 0.92. Same formula was used for
the remaining pigments thats why the group
Pavia, D. L., Lampman, G. M., Kriz, G. S., &

Engel, R. G. (1999). Organic Laboratory
echniques: A Microscale Approach(3rd Ed.).
Hardcourt College Publisher.
Robards K., Haddad, P. R., Jackson, P. E.,

(1994). Principles and Practice of Modern
Chromatographic Methods. San Diego, CA:
Academic Press Inc.
Fedessender, J. S., Fedessender R. J., & Feist

P. (2001) Organic Laboratory Techniques.
Canada:Brooks.
Williams, T. I. (1997). An Introduction to

Chromatography. New York: Chemical
Publishing Co., Inc.
Pastro, D. J., John, C. R., & Miller, M. S.

(1998). Experiment and Technology in
Organic Chemistry. New Jersey: Prentice
Hall.
Retrieved August 11, 2010

http://orgchem.colorado.edu/hndbksupport/
TLC/TLC.html
Retrieved August 11, 2010
http://www.chemguide.co.uk/analysis/chrom
atography/paper.html

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Separating Pigments of Chile Pepper Using Column Chromatography and Thin

solvent is the mobile phase; it moves up the

In the experiment, the group used column

compound is compared with the Rf of a

Figure 1. Column Chromatography set-up.

In column chromatography, the mixture to

Figure 2. TLC set-up.

The objective of the groups experiment is

The Chile peppers were triturated using the

from it is collected using a vial, covered and

plate. The components were visualized and

Using the Pasteur pipette with the cotton in

Results and Discussion

One milliliter of the extract was introduced

Figure 3. The extract from Chile pepper.

For column chromatography, as we poured

The eluates were applied on the TLC

Figure 4(left) and 5(right). Column Chromatography set

Table 1. Column Chromatography results

The volume of the yellow pigment was 100

Figure 6. Thin Layer Chromatography product.

With reference to Figure 6, after collecting

set up first because this ensures complete

got 0.81 retention factor for the dark orange

Table 2. Thin Layer Chromatography Results

After the TLC plate was dried and viewed

Also based from table 2, the Retardation

Pavia, D. L., Lampman, G. M., Kriz, G. S., &

Robards K., Haddad, P. R., Jackson, P. E.,

Fedessender, J. S., Fedessender R. J., & Feist

Williams, T. I. (1997). An Introduction to

Pastro, D. J., John, C. R., & Miller, M. S.

Retrieved August 11, 2010

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