Chromatography

Abit, Samantha T., Abrenica, Joelle Marie A., Alejandrino, Louie Aldrei S.,
Armea, Arnel Jr., Baluca, Christine Joy, and Bencito, Princess Thea B.
Group 1 2G Medical Technology Organic Chemistry Laboratory
ABSTRACT
Chromatography is a method used for separating and analyzing desired components in a sample mixture. The
objective of this experiment is to separate the components of siling labuyo (Capsicum frutescens) and malunggay
leaves (Moringa Oleifera). Column chromatography and thin layer chromatography was used in the experiment to
separate the colored components, determine the polarity and to compute the Rf value of the colored pigments. The
colored components obtained by thin layer chromatography were light yellow, yellow and orange while the column
chromatography were clear orange, peach, viscous orange, bright orange and light orange.

INTRODUCTION
Chromatography is a separation technique
based on the rates at which the components of
mixture are carried by stationary phase though a
mobile phase. The stationary phase is fixed in
place and separates the components with
different affinity by means of polarity; while,
mobile phase or moving phase moves the
component through the stationary phase.
There are different types of chromatography
which are based on the type of surface and this
involves the planar and column chromatography.
Planar
chromatography
includes
paper
chromatography and thin layer chromatography
(TLC).

Figure 2. Column Chromatography Set-up

Thin layer chromatography is used for
identifying components, determining their purity
and following the progress of a reaction. It also
permits the optimization of the solvent system
for a given separation problem[1].
Column chromatography is
preparative
technique used to purify compounds depending
on their polarity or hydrophobicity. It is based on
both solubility and adsorptivity[2].

EXPERIMENTAL
A. Compounds tested (or Samples Used)
Figure 1. Thin Layer Chromatography Set-up
Meanwhile, gas chromatography (GC) and
high performance liquid chromatography (HGLC)
comprises column chromatography.
The types of chromatography used in this
experiment were thin layer chromatography and
column chromatography.

Three pieces of red siling labuyo (Capsicum

frutescens) and malunggay leaves (Moringa
Oleifera)

B. Procedure
Column chromatography was used and it
consists of iron stand, iron clamp, pasteur
pipette, test tubes and test tube rack. (see figure
1)

1. Extraction

Three pieces of siling labuyo were cut into

pieces and the seeds were removed. It was
pounded using mortar and pestle; the pigments
were extracted using dichloromethane (DCM)hexane (1:1)

RESULTS AND DISCUSSION

Figure 3. Extraction of Siling Labuyo

Figure 5. Eluates

2. Column Chromatography
Plug cotton on the pasteur pipette and
uniformly filled it with silica gel up to the
intended part of the pipette. Put 0.5mL of the
extract on the top of the column and introduced
it
with
DCM-hexane
(1:1),
DCM
and
DCM:methanol (1:1). Collect the colored eluates
in different test tubes and note the number of
drops of colored eluate in each vial. Discard
colorless eluates.

Based on the data gathered (see Table 1),

colored pigments were successfully extracted
using
column
chromatography.
Different
pigmented eluate was collected from the three
siling labuyo and was observed to be clear
orange, peach, viscous orange, bright orange and
light orange appearance.

Color of Component

Volume of eluate
(drops)

Clear orange

28 drops

Peach

16 drops

Viscous orange

19 drops

Bright orange

8 drops

Light orange

5 drops

Table 1. Results from Column Chromatography

In
comparison
with
the
thin
layer
chromatography; light yellow, yellow and orange
was obtained from the experiment (see Table 2).
Rf value were computed and it was shown that
the farther the distance of component from
origin, the higher the Rf value. The following
formula was used for the computation of Rf value.

Color of
component
Figure 4. Column Chromatography
Light yellow
Yellow
Orange
Table
2.
Chromatography

Distance of
component
from origin
(x) in cm
4.6 cm
3.3 cm
0.9 cm
Results

from

Rf value
0.85
0.61
0.17
Thin

Layer

With the results gathered from column

chromatography and thin layer chromatography,
it was shown that both of the methods were able
to obtain the same results. However, in
comparing the rate of development, thin layer
chromatography is more efficient because it
developed faster and column chromatography is
time consuming.
REFERENCES:

[1] Definition of Thin Layer Chromatography

http://www.chem.ucla.edu/~bacher/General/30BL/tips
/TLC1.html; Date retrieved: October 26, 2016
[2] Definition of Column Chromatography
University of Toronto Scarborough. Column
Chromatography Theory.
http://www.utsc.utoronto.ca/webapps/chemistryonline
/production/column.php; Date retrieved: October 26,
2016