COLUMN CHROMATOGRAPHY

Harvie M. Barcellano, Dariene Joie B. Bautista, Florianne May D. Beltran, Jerika Prem
M. Benjamin, Rochelle Anne C. Burata and Emmanuel R. Caliwag
Group 2
2G Medical Technology
Organic Chemistry Laboratory
ABSTRACT
Column chromatography is a method used to purify individual chemical compounds from mixtures of compounds. It is
a useful method for separating solids (stationary phase) and liquids (mobile phase). Siling labuyo (Capsicum
frutescans) is a small, conical, and red cayenne pepper commonly found in Southeast Asian countries. The experiment
aimed to separate the colored components of siling labuyo using column chromatography. In this experiment,
pigments of the siling labuyo were extracted with the use of DCM-hexane, DCM, and DCM-Methanol as eluents. Crude
extract of siling labuyo was added to a column (Pasteur pipette filled with silica gel) and eluents were introduced in
succession. Three colored eluates were collected namely, 75 drops of yellow pigment (least polar), 73 drops of orange
pigment and 81 drops of yellow-orange pigment (most polar).

INTRODUCTION
Chromatography is a technique for
separating the components, or solutes, of a
mixture on the basis of the relative
amounts of each solute distributed between
a moving fluid stream, called the mobile
phase, and a contiguous stationary phase.
The mobile phase may be either a liquid or
a gas, while the stationary phase is either a
solid or a liquid.2 Various types of
chromatography are possible, depending on
which two phases are used. These are solidliquid (column, thin layer), liquid-liquid
(paper, high-performance liquid), and gasliquid
(vapor-phase)
chromatographic
methods.1
Column chromatography is one of the
most useful methods for the separation and
purification of both solids (stationary phase)
and liquids (mobile phase). 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). The
solid may be almost any material that does
not dissolve in the liquid phase. The most
common solids used are silica gel and
alumina.1
Chili pepper (Capsicum frutescans) or
siling labuyo is a variety of cayenne pepper
found in Southeast Asian Region with strong
pungent or hot taste. Its fruit is small,
conical and tapering in shape and about 1.5
to 3 cm long. Siling labuyo (Capsicum
frutescens) may be pale green or yellow
turning red as it ripens and contains many
small seeds.3 Its active ingredient is
capsaicin (Figure 1), a vanilloid, which is
responsible for the spiciness of chilis. It
causes a burning sensation when it comes
into contact with mucous membrane, due to

its interaction with pain and heat sensing

neurons.4
This experiment aims to separate the
colored components of siling labuyo
(Capsicum
frutescens)
using
column
chromatography.

EXPERIMENTAL
A. Samples Used
7 pieces of red siling labuyo (Capsicum
frutescens), DCM-Hexane (1:1 ratio), DCM,
and DCM-Methanol

B. Procedure
1. Extraction of siling labuyo
The stems and seeds of siling labuyo were
removed. These were then cut into fine thin
pieces and were subjected to trituration.
DCM-hexane was added to the triturated
siling labuyo and a dark-orange liquid
extract was obtained. It was separated to a
test tube using a Pasteur pipette.
2.
Preparation
of
column
chromatography set-up
A Pasteur pipettes column was plugged
with cotton and was packed uniformly with
silica gel up to its indented part. An iron
clamp, securely attached to the iron stand,
was used to hold the column (shown in
Figure 2).
3. Column chromatography of siling
labuyo extract
20 drops (approximately 0.5 mL) of
extract was placed on top of the Pasteur
pipettes column. Three eluents were added
in succession, as follows:

First, DCM-hexane was introduced in

small quantities in the column immediately
after placing the extract.
Second, DCM was introduced after the
transition of colors.
Lastly, DCM-methanol was placed after
the next transition of colors.
The colorless eluates were discarded. The
number of drops of each colored eluate
collected was counted.

RESULTS AND DISCUSSION

Separation occurs when an eluent of low
to moderate polarity is passed through the
column. Less polar compounds of the
mixture readily dissolve in the eluent and
move through the column. More polar
compounds have a stronger attraction to
the adsorbent than to the moving eluent.
When differences in attraction are great
enough, the compounds can be separated.
On the addition of DCM-hexane, a yellow
eluate was obtained. Next, DCM was
introduced and an orange eluate was
collected. Lastly, a yellow-orange eluate
was collected upon the addition of
DCM:methanol.
Therefore, the yellow-orange eluate is the
most polar compound, followed by the
orange eluate and the yellow eluate is the
least polar compound.
The third eluate (yellow-orange) has a
greatest volume with 81 drops, followed by
the first eluate (yellow) with 75 drops and
lastly, the second eluate (orange) with 73
drops.
Table 1. Eluates
Chromatography

collected

Color of Compound
1
2
3

Yellow
Orange
YellowOrange

in

Chemistry Revised Edition. 839 EDSA,

South Triangle, Quezon City: C&E
Publishing, Inc. page 57
[2] Keller, R.A. Chromatography.
http://www.britannica.com/science/chroma
tography. Retrieved last October 21, 2015.
[3] Siling Labuyo- Herbal Medicine.
(2011).Retrieved October 21, 2015, from
http://www.medicalhealthguide.com/article
s/silinglabuyo.htm#d
[4] VanAlphen, B.(2015).Why Chilli Peppers
are Spicy: The Chemistry of a Chilli.
Retrieved
October
21,
2015,
from
http://www.compoundchem.com/2014/01/
15/why-chilli-peppers-are-spicy-thechemistry-of-a-chilli/
Figure 1. Capsaicin

Figure 2. Column Chromatography SetUp

iron stand

column
iron clamp

Column

test tubes
for the
collection
of eluates

Volume of
eluate (drops)
75
73
81

These eluates represent the colored

pigments found in red siling labuyo which
are yellow, orange, and yellow-orange
(Figure 3).

REFERENCES
[1] Bathan, G.I., Bayquen, A.V., Crisostomo,
A.B.C., Cruz, C.T., de Guia, R.M., Farrow,
F.L., Pea, G.T., Sarile, A.S., & Torres, P.C.
(2014). Laboratory Manual in Organic

Figure 3. Colored Eluates Collected