Column Chromatography of Siling Labuyo

Yya Cabauatan, Denise Caramoan, Christine Cerrero, Christopher Cipriano, Joshua Adrian Crisostomo,
Joseph Cua
Group 2, 2F-PH, Department of Pharmacy, Faculty of Pharmacy
Abstract
Chromatography is a technique used to separate components of a mixture. Like any other
technique, chromatography has different types, one of which contain an advantage and disadvantage
over the other. In this experiment, we used DCM hexane to extract the pigments of siling labuyo, this
being the column chromatography. Later on, the components of the extract was tested using thin layer
chromatography. UV light was then utilized to see the developed TLC plate. Retention or retardation
factor was then measured.
Introduction
Chromatography is a way of separation of a mixture between two phases. The stationary phase
remains in the same position while the mobile phase moves through the medium carrying its
components. The components are slowed down in movement in comparison to the mobile phase by the
stationary phase, which acts to constrain said components from moving. Furthermore, chromatography
utilizes the differences of the phases in terms of solubility or absorptivity that is classified whether being
stationary or mobile phase.
Chromatography is mainly used for analytical use or purpose. In the right conditions, it can be
used for a preparative scale separation in which three elements are considered being: load, resolution,
and speed. It is ideal that all of these elements be maximized so that the results could be quickly
achieved with efficiency. However, it is more often than not that only two are maximized with the
expense of one element. Often, for routine work, the resolution and speed are the ones maximized and
the load taking all the expense, but in a preparative scale separation, the load and speed are maximized,
but this results to incomplete separation of the components. Complete separations are achievable only
that it takes time and is drearier to do.
Column chromatography and thin layer chromatography were used in this experiment. The
advantage of column chromatography over other types of chromatography is that it can be applied in
analytical and preparative uses. It is used to determine the number of components and to separate and
purify quantities of those components for analysis. However, there is also a disadvantage in using this
method, it being that it is time and effort consuming. If it is used for analytical purpose, paper
chromatography could be substituted.
Thin layer chromatography, on the other hand, is a method that uses a strip or plate of glass with
an adsorbent side. The solvent then travels up the plate with the help of capillary action. This method
has advantages, some of which are the following: inexpensive, simple, quick, and this only requires a
small amount of the samples. This is generally used in analysis, determining the purity or number of
components of the said mixture. It is also used to know the best solvents to be used for a column
chromatography separation.
Dichloromethane (DCM) hexane is the solvent system utilized to elute through the column. The
mobile phase consists a 1:1 ratio of DCM (CH2Cl2) and hexane (C6H14). The solid phase used was the
silica gel. It is then purified and eluted with solvent system until the compound that is desired comes out
of the column.

Rf value refers to the ratio of the distance traveled by the spot relative to the moved by the
solvent, in this experiment, the DCM hexane.
Rf=distance travelled by compound/distance travelled by solvent
The goals of the experiment is to separate the colored components by means of column
chromatography and to test for purity using TLC or thin layer chromatography ; measure the Rf of
components in TLC.
Methodology
The siling labuyos pigments were extracted by means of pounding in the mortar and pestle with
the aid of the DCM-hexane to help get the extract. The extract was then collected to be used in the next
steps of the experiment.
A setup was made so that a pipette was attached onto an iron rod. It was later filled with silica
gel that was first plugged with cotton. The silica gel was packed uniformly so that no air bubbles are
made until it made it up to the indented part of the pipette.
The extract being 0.5ml in volume was placed on the top of the column with the use of a pipette.
It was later eluted using 10ml of DCM hexane. The system solvents were poured in portions to avoid
overflowing at the top most part of the column. At the moment that it was about to run dry of the
solvent, more is added because the column cannot be allowed to dry up. Colorless eluate was collected
and discarded. The test tubes that collected the eluates were changed as the colors changed. The number
of drops of each eluate was recorded. After the column chromatography was performed and eluates
completely collected, experiment proceeded to performing the TLC.
The eluates were then put on the pre-coated TLC plate that measured at 5cm x 8cm, equidistantly
spotting them and making sure that they were as small as possible so that they wouldnt disarray. The
spot(s) were allowed to dry first before adding the next spots.
An approximate amount of DCM hexane was placed into the developing chamber that was lined
with filter paper for the plate to stand on. It was then covered with a watch glass and set equilibrate.
After that, the developing plate was then put in the developing chamber carefully. The solvent system
was allowed to be incorporated and rise up until it reached about 1cm before the upper end. The plate
was then removed carefully from the chamber and solvent front was marked and allowed to dry.
It was later then subjected to a UV lamp so that the components could be visualized, and Rf
values be measured and plate be recorded.
Results and Discussion
Sample: Siling Labuyo
Solvent System: DCM Hexane
Column Chromatography
The extraction yielded to 3 different colors of pigments from the siling labuyo extract. These
being red, yellow, and orange. The volume of the eluates were equal, amounting to about 4 drops of
each eluate.

Figure 1: Developed TLC plate viewed under regular lighting setting

UV lighting was used in viewing the developed colors in the plate that is shown in Figure 1. The
distance traveled by each of the spots were measured and plotted on a table to be used in computing the
Rf. Earlier, it has been discussed that the Rf also known as the Retention or Retardation Factor, is the
ratio of the distance travelled by the compound and distance travelled by the solvent. Therefore, since
this is a ratio, the Rf has no unit.
Computation for the Rf is shown below together with the table with the distance of the solvent
being 6cm.
Color of the Component

1
2
3
4

Red (Crude)
Yellow
Orange
Red

Table 1: Table of Results from TLC

Crude = 1.4cm = 0.2

6cm
Yellow = 0cm = 0.0
6cm
Orange= 3.5cm = 0.58
6cm
Red = 1.5cm = 0.25
6cm

Distance of
Component from
Origin(x) in Cm
1.4 cm
0 cm
3.5 cm
1.5 cm

Rf Value

0.23
0.0
0.58
0.25

The separation of the colors were not shown completely. Errors couldve come from the TLC
plate spotting or from the process in which the plate has been subjected to. Another is that the chamber
was not sealed or covered enough for the full development of the plate, resulting into a somewhat
erroneous result in the end.
References
Websites:
Chromatography
http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography
Column Chromatography
http://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.html
Thin Layer Chromatography
http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.html