DETERMINING THE COMPONENTS OF RED SILING LABUYO THROUGH COLUMN AND

THIN LAYER CHROMATOGRAPHY

Christine Joy de Gucena, Nicole Trishia Deparine, Marion Dimayuga, Angelica Gabrielle Gabinete, Jean
Ira Garcia, Lesley Bernadette Gomez
2A, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Chromatography is the most useful and modern means of separating mixtures for purification and
identification of components. All types of chromatography consist of a stationary phase, fixed medium,
and mobile phase, the moving medium. Column and thin-layer chromatography were performed in this
experiment. The bases of this chromatography are solubility, adsorptivity, and polarity of compounds.
Based on the results obtained, DCM-hexane (1:1) is the least polar among the eluents used and
DCM:methanol (1:1) is the most polar with the greatest distance traveled. Carotenoids, such as - and carotene, zeaxanthin, lutein and -cryptoxanthin, and anthocyanin are present in red siling labuyo.
Capsanthin is responsible for its red color.

I.

INTRODUCTION

Column chromatography is the process

where adsorption in a column process is
directly related to the affinity of the
solute for either the stationary adsorbent
or the flowing solvent. In thin-layer
chromatography, the mobile phase
ascends a thin layer of adsorbent.

II. Chromatography is used to

establish the amount of individual
components in a mixture by comparison
with suitable standards. In all
chromatographic
processes,
the
stationary phase is the medium fixed in
the system and the mobile phase is the
medium that flows through the fixed
medium. The nature of the interaction
between the components to be separated
and the two phases forms the basis for
classification. Knowledge of the
mechanism enables predictions about
the expected behavior of a system and in
choosing a stationary phase and mobile
phase combination.
III. Different
types
of
chromatography
are
performed
according on which two phases are used.
Examples are column and thin-layer
chromatography for solid-liquid phases,
paper and high performance liquid
chromatography
for
liquid-liquid
phases,
and
vapor-phase
chromatography for gas-liquid phases.

IV. The objective of this experiment

to separate the colored components of
red siling labuyo (jalapenos) leaves
using column chromatography. It also
aims to determine the purity of the
components
using
thin-layer
chromatography. At the end of the
experiment, Rf values of the colored
components in TLC are expected to be
measured.
V.
VI.

MATERIALS AND METHODS

VII.
Before extracting the
pigments of the red siling labuyo, the
seeds of the red siling labuyo were
removed in order to lessen its spiciness.
Four fresh red siling labuyo were

crushed using the mortar and pestle.

Enough
dichloromethane
(DCM)hexane with a ratio of 1:1 was used as
the extracting solvent. (See Fig. 1) A
few drops of the extract were set aside
for TLC. For the set-up of column
chromatography, cotton was plugged
into the column (Pasteur pipette). Silica
gel was packed up to the indented part
of the pipette. 0.5 mL of the extract was
placed on top of the column.
VIII.
When
the
extract
reached the silica gel, 3.0 mL each of
DCM-hexane
(1:1),
DCM,
DCM:methanol (1:1) were introduced
using a Pasteur pipette. Each eluent was
incorporated in such a way that the
column did not run dry. (See Fig. 2)
Three colored eluates were collected and
put into separate test tubes labeled with
the noted number of drops of each
eluate. The colorless eluate was
discarded.
IX.

Two lines were made in the 5 cm x

8 cm TLC plate for the 1 cm
measurement on the top and bottom
of the plate. The three eluates were
applied on the bottom line having
four previously dotted equidistant
dots. The eluates were spotted 10
times and the crude extract was
spotted five times aided by the
colorless end of the capillary tube.
(See Fig 3) One capillary tube was
used per sample during the spotting.
Prior to the application of eluates,
the developing chamber was
prepared. The solvent system,
DCM-hexane (1:1), was placed into
a 250-mL beaker. The chamber was
lined with filter paper and covered
with a watch glass.
X. After allowing the filter paper to
equilibrate, the TLC plate was placed in
the developing chamber. (See Fig. 4)
When the solvent system rose up to the
previously lined upper line, the plate
was removed. The solvent front was

marked and the plate was air-dried. UV

lamp was used to visualize the
components. To compute for the Rf
value of the components, this formula
was used;

Rf =

XI.

XII.
traveled.

danalyte
dsolvent
wherein, d is the distance

XIII.
XIV.

RESULTS AND DISCUSSION

XV.In the separation of the colored

components of red siling labuyo using
column chromatography, silica gel was
used as an adsorbent. Adsorbent is a
porous material that can suck up liquids
and solutions. Since the adsorbent used
is packed in a vertical column, there is
no need for any binders. The particle
size and size distribution directly affects
the flow of the solvent. The graph below
shows the number of drops obtained
from each colored eluate.
XVI.

Volume of eluate (drops)

160
140
120
100
80
60
40
20
0

Volume of eluate (drops)

XVII.
Eluate is the solution
obtained by eluation while eluent is the
portion of the mobile phase that carries
the components with it. Errors such as
less extract placed than the desired
amount, allowing the column to run dry,

and incorrect counting of drops collect

may have caused the inconsistent
volume of eluates. The addition of
DCM-hexane produced a yellow eluate
while DCM produced an orange eluate
and DCM:methanol (1:1) produced a
dark orange to red eluate. The eluates
represent the colored pigments found in
red siling labuyo which are yellow,
orange, and red.
XVIII.
Chromatography
is
frequently used to confirm either the
presence of absence of a compound in a
sample. The number of spots in thinlayer chromatography indicated the
minimum number of components. The
purity of a compound can be checked
and the presence of a single spot is taken
as an indication of purity. In this
experiment, minimum of one spot per
eluate manifested. Both in the first and
second eluate, there is only one visible
color but in the third eluate there are
separate yellow and orange spots. On
the other hand, the crude extract showed
several pigments with different distance
traveled from the origin. The position of
an analysis on the TLC plate is
characterized by the retention factor Rf.
The Rf value varies between 0 (analyte
remains on the start) and 1 (analyses
move with the front of the mobile phase.
(See Fig. 5)
XIX.
The
first
pigment
produced by the first eluate traveled 6.9
cm while the longest distance of the
pigment from the crude extract travele
7.0 cm. The proximity of these two
accounts to the eluent used since DCMhexane (1:1) was the first to be
introduced into the column and the
extracting solvent used on the first
procedure is DCM-hexane (1:1) as well.
These two favored the mobile phase
which consists of the same solvent.
Also, the distance traveled of the
pigment of the light orange eluate is
close to the distance traveled by the
orange eluate. The polarity of DCM and

DCM:methanol (1:1) may be the reason

behind this.
XX.
The eluents used have
increasing polarity caused by the
functional groups present. DCM-hexane
is the least polar among the three
because the non-polarity property of
hexane affects the polarity of DCM and
DCM:methanol is the most polar
because methanol is an alcohol which
has high polarity. Red siling labuyo or
ripened jalapenos is a cultivar of the
species Capsicum annuum. Chili pepper
fruits undergo metabolic transformations
in terms of pigment composition.
Anthocyanins produce purple pigments,
and the yellow-orange colors are
provided by - and -carotene,
zeaxanthin, lutein and -cryptoxanthin.
Capsanthin is a red color pigment that is
lipophilic and is mostly synthesized and
accumulated in red Capsicum spp. Refer
to Figure 6 and Figure 7 for the
molecular structure of the pigments
present in red siling labuyo.
XXI.
8
7
6
5
4
3
2
1
0

XXII.
XXIII.
XXIV.
XXV.
XXVI.
XXVII.

Distance of
component
from origin
(cm)
Rf value

XXVIII.
XXX.
XXXI. REFERENCES
XXXII. Colombini, M., & Modugno, F.
(2009). Organic Mass Spectrometry
in Art and Archaeology. West
Sussex, United Kingdom: John
Wiley & Sons, Ltd
XXXIII.
XXXIV.
Cserhati, T. (2007). Liquid
Chromatography
of
Natural
Pigments and Synthetic Dyes.
Amsterdam,
The
Netherlands:
Elsevier
XXXV. Robards, K., Haddad, & P., Jackson
P. (1994). Principles and Practice of

XXIX.
Modern Chromatographic Methods.
London,
United
Kingdom:
Academic Press Limited
XXXVI. Shugar, G., & Ballinger, J.
(1990).
Chemical
technicians
Ready Reference Handbook (3rd
Ed.). USA: McGraw-Hill, Inc.
XXXVII. Solvent Miscibility and Polarity
Chart. Retrieved September 22,
2014
from
http://www.perfinity.com/downloads
/Solvent%20Miscibility%20and
%20Polarity%20Chart

XXXVIII.
XXXIX.