Column Chromatography of

Pigments from Capsicum

frutescens
ARCANGEL, Nicholas
CUAN, Lindane
GONZALES, Kym
MESA, Hannah
SO, Ignacius

I.Introduction: Capsicum frutescens

Capsicum frutescens (siling labuyo) is a kind of pepper commonly
found in Southeast Asia. It is mistaken as Capsicum annum which
has similar physical characteristics but siling labuyo has a greater
pungent odor and taste.
Siling labuyo has therapeutic actions such as vasomotor stimulant,
aphrodisiac, pain reliever and many more.

I.Introduction: Capsicum frutescens

Pigments are molecules that absorb light energy in the visible
range. The color we see is the color that is not absorbed but is
reflected.
Major Pigments present in Capsicum frutescens:
Carotenoids- helps in light absorbstion for photosynthesis
Xantophylls- accesory pigments.

Introduction: Capsicum frutescens

- major red colored pigment: capsanthin

I.Introduction: Capsicum frutescens

-minor red colored pigment capsorubin

I.Introduction: Capsicum frutescens

-yellow pigment Beta-carotene

I.Introduction: Chromatography
widely used method for the separation, identification, and
determination of chemical components in complex mixtures by
distributing them between two phases:
1.Stationary phase (adsorbents) - phase that is fixed in
place either in column or on a planar surface (silica gel as
adsorbent)
2.Mobile phase (eluents) - phase that moves over or through
the stationary phase, carrying with it the components of the
mixture (hexane-dichloromethane-methanol as eluting
solvent)

I.Introduction: Column
Chromatography
In Column Chromatography, the stationary phase (silica gel) is held in a
narrow tube (Pasteur pipette), and the mobile phase is forced through the
tube under pressure or by gravity. It is generally used as a technique to isolate
desired compounds from a mixture.
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.

I.Introduction: Silica gel

glass-like, porous structure of SiO2
the pores create a huge surface area which
will bind water through surface adsorption
and capillary action
adsorption - the binding of molecules or
particles to a surface, must be
distinguished from absorption, which
is the filling of pores in a solid

I.Introduction: Eluting solvents

1.Hexane (C6H14)

2.

Dichloromethane

(DCM) (CH2Cl2)

3.

Methanol

(CH3OH)

alkane
nonpolar
halogenated hydrocarbon
semi-polar

alcohol
polar

II. Objective
To separate the coloring components
(pigments) of Capsicum frutescens
using column chromatography.

III. Materials
5 pieces chili peppers or siling labuyo (Capsicum frutescens)
Silica gel (SiO2)
Dichloromethane (DCM) (CH2Cl2)
Hexane (C6H14)
Methanol (CH3OH)
Pasteur pipette

Beaker

Cotton plug

Scissors

Sand

Mortar and Pestle

IV. Set-up

V. Methodology
A.Preparation of the samples
5 pieces chili peppers
1.Remove the seeds.
2.Cut into small pieces.
3.Triturate with 2 tablespoons of sand and 5 ml DCM.
Homogenized sample

V. Methodology
B.

Packing of the column

Pasteur pipette
1.Clog with small cotton.
2.Add silica gel until of the Pasteur pipette is
filled.
3.Secure with clamp.
Packed column

V. Methodology
C. Column loading
Packed column
1.Add 15-20 drops of homogenized
sample.
2.Add 5 ml eluting-dropwise solvent.
3.Use clean test tube to collect the
eluent.

Eluting Solvents
1. Hexane
2. Hexane-DCM (1:1)
3. DCM
4. DCM-Methanol (1:1)
5. Methanol

VI. Data and Results

VI. Data and Results

VII. Discussion

VII. Discussion
Three major bands of colors were eluted: yellow, light orange, and
red but the intensity of each color in relation to each eluent differs.
There are notable differences regarding the sequence of the bands of
color that each group got. Further research was made and the
findings are that the most non-polar component pigment was the
yellow band (Beta-carotene), being the first to be eluted from the
column, because hexane was first used which is a non polar eluting
solvent.

VII. Discussion
The color of the second eluent was a combination of yellow and
orange due to the Hexane:DCM solvent. For the third eluent, DCM,
the resulting color was dark orange. A fourth and a fifth eluent were
also used, but one of the groups were not able to reach these due to
how the setup was prepared. However, for the groups that were able
to, the colors that were produced were darker than the preceding
eluents.

VIII. Conclusion
The major component pigments of Capsicum frutescens can be
separated by the usage of column chromatography. The separation of
components were explained on the basis of adsorptivity, polarity and
solubility of the sample and the solvents used.

IX. References
Brown, W. H., Foote, C. S., Iverson, B. L., & Anslyn, E. V. (2011).
Organic chemistry (5th ed.). California, CA: Brooks/Cole Cengage
Learning.
Organic Lab. (n.d.). Column chromatography procedures. Retrieved
from
http://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Pro
cedure.html
Raven, P. H., Johnson, G. B., Mason, K. A., Losos, J. B., & Singer, S. R.
(2014). Biology (10th ed.). New York, NY: McGraw Hill Companies