UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY
Organic Chemistry Laboratory
A.Y. 2018-2019

Experiment no. 5
Column and Thin Layer Chromatography
Calugay, J.J., Caparas, C.E., Dador, A.S., De Guzman, D., & De Villa, G.K.

Abstract
Chromatography is a technique for separating components or solvents in a
mixture based on their relative affinities towards stationary and mobile phases. It aims
to identify, purify, analyse, and quantify the mixtures of a specific sample. In this
experiment, pigments of the Siling Labuyo, scientifically known as Capsicum frutescens,
were extracted through crushing and grinding, and then adding DCM-hexane. The
extract was introduced into the column and the eluate was collected. This process is
called the Column Chromatography (CC). The purity of the components was then
determined using thin layer chromatography (TLC). After which, the RF or the
Retardation factor was measured. Xanthophylls are the yellow pigments from the siling
labuyo which travelled the shortest distance, thus, they are considered the most polar
pigment. While the orange pigments obtained are called Carotenoids, which travelled
the fastest and furthest, thus, they are nonpolar because they were attracted to the
solvent which is also nonpolar rather than the TLC paper.
Introduction
Chromatography is a powerful and mobile phases, also known as
analytical method that pertains to a “differential partitioning”.
variety of laboratory techniques that are
For example, a compound that
used for the separation of mixture by
interacts weakly with the stationary
distribution through different phases,
phase would move in the mobile phase
one being stationary phase, and the
rapidly, likewise, compounds that
remaining phase being mobile phase.
interact with stationary phase strongly
The various constituents of the mixture
will move slowly. Thus, to ensure an
travel at different paces, causing them
ideal separation, it is favorable to have
to separate. This occurs because
different partition coefficient between
different substances have different
mobile phase and the stationary phase.
partition coefficients between stationary
As a result, the different rates would The solvent moves up the plate through
result to a complete separation. capillary action, carrying it with different
components of the mixture at different
In the experiment, column and
rates. The results were series of spots
thin layer chromatography (TLC) were
on the plate perpendicular to the
used. Column chromatography was
solvent. These spots were used to
generally used as a purification
determine the Rf values of the different
technique because it was used to isolate
components.
the desired compounds from the
mixture. On the other hand, Thin layer The objectives of the experiments
chromatography was generally used as are:
a qualitative analytic such as checking
(1) Separate the colored components
the purity of the compound or
of siling labuyo using column
determining the number of components
chromatography
in the mixture.
(2) Determine the purity of the
The eluates were applied in the components using thin layer
TLC plate by spotting 10 times using a chromatography (TLC)
capillary tube, 0.75 cm from the edge of (3) Measure the retardation factor
the plate, and then the plate was placed (Rf) value of the colored
in a developing chamber, with enough components in TLC
solvent just below the point of origin.

Methodology Pigments of red siling labuyo

were extracted by cutting it to pieces
Materials used were mortar and
and pouring DCM-hexane (1:1) into it.
pestle, Pasteur pipettes, iron stand, iron
Then, it was triturated using the mortar
clamp, beaker, watch glass, test tubes,
and pestle. The extracted pigments
capillary tubes, precoated TLC plates,
were set aside for a while.
filter paper, siling labuyo, silica gel,
cotton, dichloromethane, hexane, and Silica Gel Column was prepared
acetone. by plugging the column with cotton and
uniformly packed silica gel until it
reached the neck of the Pasteur pipette.
After which, 0.5 mL of the siling labuyo
extract was placed on top of the
column. The solvent was introduced in
succession with 3.0 mL each of DCM-
hexane (1:1), DCM, and DCM:methanol
(1:1). The column was not allowed to
run dry. The colorless eluate was
discarded while the colored eluates were

Fig 1. Obtaining the extract of red siling labuyo

collected in a separate test tube. Test Developing chamber was
tubes were changed every time the prepared by placing an approximate
color of the eluate changes. Number of amount of the DCM-hexane. The inner
drops for each color was counted and wall of the chamber was lined with filter
recorded. paper, covered with a watch glass, and
was allowed to equilibrate. Then, the
plates were carefully placed in the
developing chamber. The solvent
system was allowed to rise up to 1 cm
from the upper end. The plates were
removed from the chamber; the solvent
front was immediately marked and was
air-dried.

Fig 2. Column chromatography setup

After collecting the eluates from

the column, Thin Layer Chromatography
was performed. The eluates were
applied on the precoated TLC plate by
spotting it 10 times using a capillary
tube. Each spot was allowed to dry Fig 4. Thin Layer Chromatography setup
before applying the next one and was
The spots were measured from
ensured that they were applied as small
the line of origin up to the middle part
as possible so that the colors would not
of the eluent. Distance traveled were
spread.
recorded and was used for the
computation of the Rf values.
Results
Column Chromatography
Upon extraction of the siling
labuyo, a dark orange extract was
obtained. Then, three solvent systems
were used in order to yield various
Fig 3. Yielded eluates
eluates. And the data collected during
the column chromatography are the
volume (in drops) of the three eluates 3 yellow 2.50 cm
obtained, which are: light or pale
yellow, yellow, and orange respectively. 4 orange 2.70 cm
Table 2. Measured distance travelled of the eluates
Color of Volume of
component eluates (drops) Based on the distance travelled
by each eluates and the distance
1 dark orange 52 drops
travelled by the solvent; which was 6.20
2 light/pale yellow 34 drops cm, their retention factor were
computed. With the formula:
3 yellow 36 drops
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒕𝒉𝒆 𝒔𝒐𝒍𝒖𝒕𝒆
Rf value=
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒕𝒉𝒆 𝒔𝒐𝒍𝒗𝒆𝒏𝒕
4 orange 48 drops
Table 1. Volume (in drops) of the collected eluates Color of Retention factor
component
With the extraction of siling
labuyo, 52 drops or 2.6mL of dark 1 dark orange 0.39
orange extract was obtained. With the
use of the three extracting solvents, the 2 light/pale yellow 0.00
first eluate yielded was light or pale
3 yellow 0.40
yellow, which was equivalent to 34
drops or 1.7mL. Then, 36 drops or 4 orange 0.16
1.8mL of yellow eluate was also Table 3. Retention factor of the collected eluates
obtained. And lastly, 48 drops of orange
Computations:
eluate was yielded, which was
equivalent to 2.4mL. Dark orange eluate
Thin Layer Chromatography Rf value=
𝟐.𝟐𝟐 𝒄𝒎
= 0.39
𝟐.𝟐𝟐 𝒄𝒎
The distance travelled by each
Light or pale yellow eluate:
eluates from the origin were observed,
measured, and recorded in order to 𝟐.𝟐𝟐 𝒄𝒎
Rf value= = 0.00
𝟐.𝟐𝟐 𝒄𝒎
compute for the retention factor or Rf
value. This were all done with the use of Yellow eluate
a precoated Thin-layer Chromatography 𝟐.𝟐𝟐 𝒄𝒎
plates or TLC plate. Rf value= = 0.40
𝟐.𝟐𝟐 𝒄𝒎

Orange eluate
Color of Distance of
component component from 𝟐.𝟕𝟎 𝒄𝒎
origin (X) in cm Rf value= = 0.44
𝟐.𝟐𝟐 𝒄𝒎

1 dark orange 2.40 cm

2 light/pale yellow 0.00 cm
Discussions
 Column chromatography was that yellow pigments or xanthophylls are
used to extract pigments from red siling polar molecules. On the other hand, the
labuyo. DCM- hexane with 1:1 ratio is orange colored eluates are able to travel
first poured will triturating 5 pieces of the farthest and the fastest from the
sili and was set aside thereafter. origin which makes it a least polar and
In a pasteur pipette, cotton was more non- polar molecule.
placed in the neck of the pipette topped
References
by silica gel. This set-up is called the
silica gel column. The siling labuyo Bathan, et al. (2018) Experiment 5:
extract was mixed with 3 solvent Column and Thin Layer
solutions with 3.0 mL each of DCM- Chromatography. Laboratory
hexane (1:1), DCM, and DCM:methanol Manual in Organic Chemistry
(1:1). The first solvent mixture (revised edition).
containing DCM-hexane solvent was
used in the silica column which yielded Bagsican, M., et al. (2018) Experiment 5:
the first eluate that is, 34 drops or Column and Thin Layer
1.7mL of light yellow solution. The Chromatography.
second containing DCM only yielded 36
Delos Santos, A.G., et al. (2016). Column
drops or 1.8mL of yellow colored eluate.
And the third which contained DCM- and Thin Layer Chromatography.
methanol gave a result of 48 drops or Retrieved
2.4mL.of orange eluate. http://courses.chem.psu.edu/che
m36/Experiments/PDF's_for_techn
The thin layer chromatography is
iques/CC.pdf. Retrieved on March
performed after collecting the eluates
24, 2019.
from the column chromatography. Using
a capillary tube, the siling labuyo
extract, and the eluates were placed
apart on a TLC plate. The TLC plate was
partly submerged in a DCM- hexane
solution until the colors from the eluates
reach a certain height. The distance
travelled by the dark orange colored
eluate or the siling labuyo extract was
2.40 cm, the slightly yellow colored
eluate did not travel at all having 0 cm
travelled, the yellow colored eluate
travelled 2.50cm and the orange colored
eluate travelled 2.70cm.
Based from the results given, the
yellow colored pigments are able to
travel least. From this, we may conclude