Separation of pigments of SilingLabuyo using Thin Layer and Column Chromatography

Miguel, K.D., Moron, R.S.S., Pazon, A.D., Ramirez, C.V., Raquepo, T.M.R., Razon, D.N.A.Jr.
2B-PH, Group No. 6, Department of Pharmacy, Faculty of Pharmacy,
University of Santo Tomas, Espaa Boulevard, 1015 Manila, Philippines
Abstract
The report is about obtaining and separating different pigments using chromatography.
Chromatography is a laboratory technique used to separate components of a mixture. There are
many types of chromatography but thin layer and column chromatography was used in the
experiment with the use of DCM-Hexane (1:1). The objective of this study is to show the
pigments present in SilingLabuyo in a chromatography paper using the thin layer method.
Caution was used because the materials used posed harmful effects to the students/researchers.
The pigments were extracted using trituration and they were separated using different solvents,
then they were shown using chromatography.

Introduction
[1]Different pigments exist in Siling
Labuyo, and one of the possible methods for
separating
them
is
thin
layer
chromatography.
In
thin
layer
chromatography a solvent system is placed
in
a
coverable
container,
the
chromatography paper which contains the
different pigments is placed inside the
container. To obtain the pigments, Siling
Labuyo was first triturated using mortar and
pestle together with a 1 to 1 mixture of
DCM and hexane, the solution produced
contains all the pigments mixed, and this is
where thin layer chromatography comes in,
to separate these different pigments. Thin
layer chromatography is used because it is
the cheapest and easiest way to separate
these pigments.
The solvent DCM used is a
carcinogenic substance, that is why anyone
who will try to do the same experiment in

the future shall not expose the solvent

frequently to themselves to avoid health
complications, also SilingLabuyo releases
irritant chemicals through the air and
through contact when opened up that can
cause inflammation in the skin and eyes
when touched. Also Hexane is a flammable
substance that is why no flame was used in
any part of the experiment. Exposure to any
of these substance may cause serious harm
to the person.
The first objective of this study is to
show the idea of how chromatography,
specifically thin layer chromatography
works. Also another purpose is to extract
and show the different pigments present in
SilingLabuyo.Also the experiment teaches
the students how to be careful and how to
properly extract the pigment even if it is an
irritant.

Methodology

[2]The general formula for the Rf values is:

distance travelled by the solvent

distance travelled by compund

Rf =

Apparatus and Reagents

The different materials used were
mortar and pestle, Pasteur pipettes, iron
stand, iron clamp, beaker, vials, capillary
tubes, TLC plates, filter paper, watch glass,
SilingLabuyo, DCM, hexane and silica gey.
Precautions
Hexane is a fuel and it shall be
placed and stored way from flame to avoid a
fire. DCM is a suspected carcinogen,Siling
labuyo is an irritant that may irritate the skin
and eyes.
Procedure
First, the pigments were extracted
using a mortar and pestle. The siling labuyo
was mixed with a proper amount of DCM
and Hexane (1:1), this solution helps on the
dissolution of the solid pigments present in
the pepper. A column was filled with cotton
and silica gel was placed on top of it, this
shall serve as the filer/ separator for the
different pigments in the extracts. 0.5 ml of
the extract was placed on the column, next
3.0ml of DCM-hexane (1:1), DCM, and
DCM-methane (1:1) was placed. The
transparent eluate was discarded and the
colored eluates were kept separately, the
number of drops was counted, they were
placed on different vials. The pigments were
spotted on the TLC plates 10 times. The
developing chamber was a 500ml beaker
filled with adequate amount of DCMHexane (1:1), it was later covered with a
watch glass and the solvent system was
allowed to nearly touch the top of the TLC.
The TLC was recovered and the data was
recorded.

Results and Discussion

[5]Eluates were produced using column
chromatography. The eluates were collected
in separate vials and recorded. Below is a
table showing the data collected.
1
2
3

Color
Yellow
Orange
Light
Orange

Volume(drops)
85
76
73

The table shows that the unknown Yellow

pigment has the highest volume (85),
Orange (76) comes in second, and Light
Orange (73) comes last.
[4]Using thin layer chromatography, the Rf
values and movement of the separate
pigments were shown and recorded

{1}https://www.scribd.com/doc/65258518/

Paper-Chromatography-Formal-ReportORG-chem
{2}http://www.ce.gxnu.edu.cn/organic/net_

course/content/tlc/Retention_Factor.htm
{3}http://orgchem.colorado.edu/Technique/

Procedures/TLC/TLC.html
{4}http://www.chemguide.co.uk/analysis/c

hromatography/thinlayer.html
{5}http://orgchem.colorado.edu/Technique/

Procedures/Columnchrom/Columnchrom.ht
ml

[3]The table below shows the distance of

component from the origin in centimeters
and the Rf value of each pigments.
Color

1
2
3
4

Crude
extract
Yellow
Orange
Light
Orange

Distance of
component
from origin in
cm (X)
0.3

Rf value

2.6
0.2
0.2

0.3611
0.0277
0.0277

0.04166

As shown above, the yellow pigment

traveled the farthest, followed by the crude
extract, orange and light orange.
References
Retrieved October 17 2015