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ORGANIC

CHEMISTRY
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Organic Chemistry Laboratory - CH 200 (2014 - 2015) Experiment 1

Extraction of Pigment of Capsicum frutescens using Column Chromatography

Anndrey M. Role, Emanuelle Rosales, Marielle San Miguel, Mikee Sison*
Department of Biological Sciences, College of Science
University of Santo Tomas, Espana Street, Manila 1051
September 4, 2014

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Abstract:
Column Chromatography is a form of solid-liquid adsorption chromatography and depends on the essential principles
as does in thin layer chromatography. It was used in this experiment in separating and analyzing the different components of
Capsicum frutescens (siling labuyo) with the use of solvents such as Hexane (C6H14), Dichloromethane (CH2Cl2),
Hexane/Dichloromethane (1:1), and Dichloromethane/Methanol (MeOH) (1:1). The components found in the experiment
were carotenoids (carotenes and lutein), xanthophylls (capsanthin) and anthocyanins, and each component varies in color
and in volume.

Keywords:
Column chromatography, adsorption, pigment, mobile phase, stationary phase, adsorbant, eluate,
eluent, analyte
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Introduction
The word 'chromatography', formed from the Greek word 'Khroma' meaning colour and 'graphein'
meaning to draw a graph or to write, was coined by the Russian botanist M.S.Tswett around 1906, to
describe his process of separating mixtures of plant pigments. The general principle employed in all kind
of chromatography is movement of a mixture of compounds over a mobile phase, which passes over
selectively adsorbing surface, the stationary phase. Two different techniques have been employed in this
experiment to separate and study the components present in spinach leaves.
Column chromatography is devised on the basis of differential adsorbance of substances on solid
adsorbent (silica or alumina) to an extent that depends on the substance polarity and other chemical
properties and structural properties. Some compounds adsorb more strongly to the stationary phase than
others, as they elute (wash down) the column at a very slow rate. Thus they can be separated on the
basis of their elution rate accordingly. This technique is used to separate naturally colored components
of leaves such as sili ( Capsicum frutescens).
Capsicum frutescens is a red pepper, siling labuyo thatis a rich source of vitamin A and C, have
antioxidants, the antioxidants combat the free radicals in the body and treat cardiac diseases, cataract,
bronchial asthma and osteoarthritis. It is great for metabolism.

A plant pigment is any type of coloured compound produced by a plant. A chemical compound
which absorbs visible radiation between 380 nm (Violet) and 760 nm (ruby-red) is considered as a
pigment. The energy from these radiations is used by plants during photosynthesis for the production of
glucose from CO2 and H2O. Different types of plant pigments exist in nature and include various classes
of organic compounds (xanthophyll, carotenoids, lycopene etc). Plant pigments give colour to leaves,
flowers and fruits and are also important in controlling photosynthesis, growth and development.
Pigments that can be found: carotenoids (carotene and lutein), xanthophylls (capsanthin), and
anthocyanins.

Methodology

Firstly, the five pieces of red chili pepper were deseeded and cut into small pieces for easy
trituration in the mortar and pestle with sand after it was weighed. After triturating, the red chili pepper
extract was obtained using decantation. The decantate was filtered in a glass funnel containing
anhydrous Sodium sulfate which is used as an inert drying agent for organic solutions; Na2SO4 is added
to the solution until the crystals no longer clump together. After filtration, the filtrate was transferred in a
100ml beaker and was mixed with silica gel and was mixed until powdery. The silica gel was used for
the pepper extract to be absorbed in it. When the extract is already powdery, it was then loaded in a
silica gel microcolumn with cotton at the end of it for plugging, sand was added to the top of the column
so that when solvent was poured into the column, it only disturbs the sand layer, and leaves the silica gel
layer (stationary phase) intact. Silica gel was also added in the microcolumn because it acts as polar
adsorbent. It was then eluted with different eluting solvents which are Hexane, Hexane:DCM (1:1),
DCM, DCM:Methanol (1:1), and Methanol. Different colors of eluents that were soluble to each eluting
solvents which are yellow, red orange, light orange, light yellow, and colorless respectively were
obtained in different test tubes.

Results
Figure 1 Different colored soluble eluents.

Table 1 The color and volume of the eluents.

* The data was retrieved from Group 10.
Soluble eluent in
Hexane
Hexane:DCM (1:1)
DCM
DCM:MeOH (1:1)
Methanol

Volume (ml)
0.9
1.7
1.9
1.6
0.6

Color
yellow
red orange
light orange
light yellow
clear

Discussion
The eluents were eluted in the column by their increasing polarity, causing a series of colors formed in
the column indicating the different components. As each eluent was dropped, a different eluate was
collected by a different test tube. As the collection was finally complete the minor and major
components could be identified by their amount of volume, minor having the least volume and major
having the most volume. The polarity of the eluents contributed to the order of how they went out of the
column and also the volume produced. This is because as the polarity increases, the power of the eluter
also increases.
The compound with the higher polarity travels slower than that of the compound having a lower polarity
because it may the ability to form a hydrogen bond. It means that it must adsorb more strongly to the
silica gel than the compound having the lesser polarity. The compound having the lesser polarity spends
more of its time in the solvent and therefore washes through the column much faster.
The first to come out of the column would be hexane soluble colored eluent, DCM:Hexane soluble

colored eluent, DCM soluble colored eluent and DCM/MeOh soluble colored eluent based on the
increasing polarity. The column at one point, produced a colorless state meaning that it has already been
purified indicating to stop the procedure. Based on the data obtained, the minor component is the test
tube which has been eluted by hexane since hexane is the least polar eluent and it also produced the least
volume.
From the data obtained, the major component is the test tube which contains the substance which has
been eluted by DCM/MeOH since it is the most polar eluent and it also produced the most volume.
The plant pigments were then identified:
a. B- carotene (C40H56) yellow (least polar)
b. Capsorbin (C40H56O) Orange
c. Capsanthin (C40H5603) Red (most polar)
All the groups had different results in color or volume because of some experimental errors such as
some recorded the colorless phase, measurements were not that accurate due to some spillage, different
weighs of the sample were used, and some may not be that purified.
Conclusion
In summary, the colored components of Capsicum frutescens which are the pigments can be
separated through column chromatography. It can also be concluded that the minor and major
components produced by eluated can be determined by the amount of volume present. Meaning, that the
least volume collected is the minor component and the major component has the most abundant volume.
It can also be concluded that the minor component is the least polar, and in contrast the major
component is most polar. The compound with the higher polarity travels slower than that of the
compound having a lower polarity because it may the ability to form a hydrogen bond. It means that it
must adsorb more strongly to the silica gel than the compound having the lesser polarity. The compound
having the lesser polarity spends more of its time in the solvent and therefore washes through the
column much faster.
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