Column and Thin Layer Chromatography

Mary Coleen A. David, Eunice Mae D. del Valle, Sean Romeo B. Desagon, Maria Cauline M. Fang, Jenicca Pamela Y. Go,
Jan Chrtien M. Guillo
Group 3, 2F-Pharmacy, Organic Chemistry Laboratory, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Chromatography is a modern and sophisticated method of separating mixtures, wherein the
physical separation of different adsorbed materials is accomplished in a single adsorbent. It can be based
on the selective distribution of chemicals between a stationary and a mobile phase. The mobile phase is
allowed to pass through the system of the separated mixture while the a well-defined spot or band is
placed as a stationary phase. There are different types of chromatography and each has its own
advantages and disadvantages. In this experiment, hexane-acetone was used to extract the different
pigments of malunggay leaves. The colored components of malunggay leaves or the extract were
separated further using column chromatography. The collected compounds then underwent Thin Layer
Chromatography (TLC). TLC was used to determine the purity and for measuring the Rf values of the
colored components.

INTRODUCTION
Chromatography is a preparative
technique
that
separates
and
isolates
components for further studies. It is a technique
in which compounds in a mixture are separated
based on differing affinities between a mobile
phase and a stationary phase. Each component
of a mixture has a different partition coefficient
between the two phases and will move through a
system at a different rate. Mobile phase is a
medium used in chromatography which moves
through the stationary phase. In TLC and
column chromatography, the mobile phase is an
organic liquid. Stationary phase is a material
used in chromatography which does not move.
The mobile phase passes through the stationary
phase. The stationary phase is either a pure solid
substance such as alumina or silica or a thin
coating of liquid on a solid support or a gel.
During column chromatography,
compounds of mixtures are separated by having
it pass through a column packed with silica gel.
Silica gel was used because compounds will
usually adhere to the silica to different extents.
Xantophyll, Chlorophyll a, and chlorophyll b

are the three colored components of the eluate

of malunggay leaves.
Thin Layer Chromatography was used
for rapid analysis of organic mixtures. TLC can
be used to help determine the number of
components in a mixture, the identity of
compounds, and the purity of a compound. In
TLC the stationary phase is a spread in a thin
layer on an inert support, usually a plastic sheet
or a glass plate. The mobile phase moves
upward by capillary action. TLC method was
also used to measure the Retention or
Retardation factor (Rf value).
Separation of the colored components of
the malunggay leaves using column
chromatography, determination of the purity of
the components using TLC, and the
measurement of the Rf values of the colored
components in the TLC were the objectives met
in this experiment.

MATERIALS AND METHODS

MATERIALS
The materials used in this experiment for
malunggay leaves are the mortar and pestle, for
grinding purposes, Pasteur pipette, for accurate
measurement, iron stand and iron clamp, to
support the column, beaker, watch glass, test
tubes, to collect the different eluates, capillary
tubes, to apply the spots, precoated TLC plates,
malunggay leaves, silica gel, cotton, and
hexane-acetone (7:3).

A column was made using a Pasteur

pipette plugged with cotton and uniformly
packed with silica gel. The column was
assembled with an iron clamp and iron stand in

METHOD
The pigments were extracted by
crushing malunggay leaves with hexane-acetone
in a mortar with a pestle.

a column chromatography set up. 0.5 mL of the

extract was placed on top of the column using a
Pasteur pipette for accurate measurement. 5.0
mL of each solvent systems (hexane-acetone,
acetone, and acetone-methanol) was introduced
successively to the set up. Solvent systems were
added to separate the colored components at
different rates.
The eluates, in different colors, were
collected in different test tubes as shown below.
(Colorless eluates were discarded) Counting the
number of drops of each eluate that are collected
in each tube was done cautiously for the
experiment.

The eluates were applied on a TLC plate,

having a measurement of 5 cm x 8 cm, and
spotted ten times using a capillary tube. Hexaneacetone, a developing chamber, was then added
in the beaker and filter paper lining the inner
wall of the beaker. The beaker was covered
using a watch glass for it to equilibrate.

The solvent system was allowed to rise up

to 1 cm from the upper end before removing the
TLC plate. The components was visualized
using a UV lamp and the distance traveled was
measured in order to compute for the Rf value.

Thin Layer Chromatography

Color
of Distance
of Rf
Component
component
Value
from origin in
cm
Yellow
4.5 cm
0.75

Yellow green

2.5 cm

0.42

Green

2.6 cm

0.43

Light green

2.7 cm

0.45

The solvent front for the TLC plate is 6 cm.

distance travelled by the component

Rf = origin
distance travelled by solvent

RESULTS AND DISCUSSION

Column Chromatography
Four colors were yielded from the
extraction of the colored components of
malunggay
leaves
using
Column
Chromatography. The number of drops of each
eluate are shown in the table below.

Color of Component

Volume
(drops)

of

Yellow

15

Yellow green

54

Green

21

Light green

76

eluate

Rf value of yellow component:

Rf=

4.5
=0.75
6

Rf value of yellow green component:

Rf=

2.5
=0.42
6

Rf value of green component:

Rf=

2.6
=0.43
6

Rf value of light green component:

Xantophylls reflect yellow components
and chlorophyll reflects green components.
Chlorophyll a reflects the darker shade of green
while lighter green or yellow green for
chlorophyll b. Xantophyll is a non-polar
compound, which was confirmed using column
chromatography. Hexane-acetone is a non-polar
compound and yellow was the first eluate to
come out. Since like dissolves like, this proves
that Xantophyll is a non-polar compound.

Rf=

2.7
=0.45
6

The distance travelled by the yellow

component is the farthest in the TLC plate
compared to the other colored components. It
travelled the farthest because it is a non-polar
compound. Polar compounds stick easier to the

TLC plates. The yellow component eventually

disappeared, leaving the green components
behind.

REFERENCES:
1. Thin Layer Chromatography. Retrieved from
http://www.chem.umass.edu/~samal/269/tlc.pdf
2. Thin Layer and Chromatography. Retrieved
from

http://www.xula.edu/chemistry/documents/orgle
clab/12TLCCol.pdf
3. Bell, C. Jr., Clark, A., & Taber, D. (2001).
Organic Chemistry Laboratory. USA:
Brooks/Cole.
4. Pavla, D. Lampman, G., Kriz, G., & Engel, R.
(1998). Introduction to organic laboratory
techniques: a microscale approach. 3rd Ed.
USA: Saunders Publishing Company.