University of Santo Tomas

Faculty of Pharmacy
Organic Chemistry Laboratory

COLUMN AND THIN LAYER CHROMATOGRAPHY

OF MALUNGGAY LEAVES ( Moringa Oleifera)
Eunice Aurelle T. Basco, Ian Lindley C. Cabral, Aira Mina A. Cayago,
Jardine Mariel L. Ching, Leomariss M. Chua and Filjosh R. Cucueco
Group 2 2A-Medical Technology Organic Chemistry Laboratory

Abstract
Chromatography is a way of separating and analyzing the constituents of a mixture by passing
them through an inert stationary environment using a mobile solvent that carries them. The
two different types of Chromatography used to achieve the objectives of the experiment were
the Column Chromatography, which is a preparative technique where the eluates were taken,
and the Thin Layer Chromatography, which is a fast technique that required only a small
amount of the material. The objectives of this experiment were to illustrate the principles of
chromatographic separations, and to demonstrate how Thin Layer Chromatography and
Column Chromatography are used in Organic Chemistry. In the experiment, the 3 eluates
collected from the extract of Malunggay leaves using the process of Column Chromatography
were the yellow-green colored component (1 st eluate), which had 60 drops, the blue-green
colored component (2nd eluate), which had 52 drops, and the green colored component (3 rd
eluate), which had 40 drops. After spotting these eluates on the Thin Layer Chromatography
plate and placing the plate in a developing chamber, it was evidently seen that from the origin,
the yellow-green, blue-green, and green eluates travelled a distance of 6.80 cm, 2.50 cm, and
2.05 cm, respectively. The UV lamp was then used to visualize the TLC plate and Rf values
were computed, giving the yellow-green eluate a 0.97 Rf value, the blue-green eluate a 0.36
Rf value, and the green eluate a 0.60 Rf value.

INTRODUCTION
Chromatography, first introduced by the Russian botanist Micharl Iswett, is a separation
method that relies on differences in partitioning behavior between a flowing mobile phase,
which is a liquid solvent (or mixture of solvents) that use to carry the sample solutes under
analysis along the paper, and a stationary phase, which is the adsorbent, to separate the
components in a mixture. [1] Chromatography is a very special separation process for a
multitude of reasons: (a) separates complex mixtures with great precision; (b) can purify
basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and
operating conditions are employed; (c) separates delicate products since the conditions under
which it is performed are
not typically severe; and (d) used to separate the colored pigments in plants. [2]
In Column Chromatography, the stationary phase, a solid adsorbent, is placed in a vertical
glass (usually) column. The mobile phase, a liquid, is added to the top and flows down through
the column by either gravity or external pressure. Column chromatography is generally used
as a purification technique: it isolates desired compounds from a mixture. [3]

Thin Layer Chromatography is a simple, quick, and inexpensive procedure that gives the
chemist a quick answer as to how many components are in a mixture. TLC is also used to
support the identity of a compound in a mixture when the Rf of a compound is compared with
the Rf of a known compound (preferably both run on the same TLC plate). A TLC plate is a
sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually
silica or alumina). [4]
In order to make the technique more scientific rather than a mere interpretation by sight,
Retention Value (Rf value) was applied in chromatography. A particular compound will travel
the same distance along the stationary phase by a specific solvent (or solvent mixture) given
that other experimental conditions are kept constant. In other words, every compound (dye,
pigment, organic substance etc.) have a specific Rf value for every specific solvent and solvent
concentration. Rf values come very handy for identification because one can compare Rf values
of the unknown sample (or its constituents) with Rf values of known compounds. [5]
In this experiment, the group should be able to attain the following objectives: (1) to be able
to separate the colored components of malunggay leaves using column chromatography, (2) to
determine the purity of the components using think layer chromatography (TLC), and (3) to
measure the Rf values of the colored components in TLC.

Methodology
Experimental:
Compounds tested (or Samples used)
Leaves of Malunggay (Moringa oleifera),
Hexane: Acetone (7:3), Acetone, Acetone
Methanol (1:1)

B.

Figure 2. Pasteur pipette plugged

with cotton and packed with silica
gel

Procedure:
1. Column Chromatography
Pigments of Moringa oleifera
were extracted by grinding the
leaves with a mortar and pestle
and adding hexane-acetone (7:3).
A portion of the extracts was then

Figure 3. Pasteur pipette

plugged with cotton and packed
with silica gel and extract on top
set aside for Thin Layer
Chromatography.
After finishing the set-up, the
pigments of Moringa oleifera were
then eluted with the use of 5.0mL
of eluents hexane-acetone (7:3),
acetone-methanol (1:1) and
acetone, which were introduced in
succession.

Figure 1. Grinded malunggay

A Pasteur pipette was then

plugged with cotton for the
purpose of bed support and was
uniformly packed with silica gel up
to the indented part. 0.5mL of the
extract was then placed on top.

the solvent system into the

beaker. DCM-hexane (1:1) was
poured into the red siling labuyo
while the hexane-acetone (7:3)
was mixed with malunggay
leaves. Then, the inner wall of the
chamber was lined with filter
paper with a watch glass on top as
covering and was next allowed to
equilibrate. Afterwards, the plates
were carefully placed in the
developing chamber and was left
for awhile in order for the the
solvent system to rise up to the 1
cm mark from the upper end.

Figure 4. Hexane-acetone,
acetone, and acetone-methanol
eluents
Figure 5. Developing Chamber
The plates were removed after
from the chamber and the solvent
front was immediately marked.
The plates then were air-dried.
After the results were obtained,
the components were visualized
through the use of UV lamp. The
data attained was used to
measure the distance traveled by
the spot and utilized for the
computation of Rf values.

Figure 6. Plate exposed to UV light

The column was not allowed to

run dry. The colorless eluate was
discarded while colored eluates
were collected in separate. The
number of drops of colored eluate
in each vial was noted.

2. Thin Layer Chromatography

On a 5 cm x 8 cm precoated TLC
plate, the eluates were applied as
small as possible by slowly
spotting 10 times with the use of a
capillary tube. The careful
spotting was to make sure that
the colors would not mix. Each
spot marked then was allowed to
dry first before the next spot was
executed.
After the careful application of
the eluates, the developing
chamber was prepared through
placing approximate amount of

Blue Green

2.50cm

0.36

Green

2.05cm

0.29

Results and Discussion

Table 2. Components obtained and their
distances from the origin
The distance travelled by the solvent
was 7.00cm from the origin of the TLC plate
to the solvent front; the first component
travelled 6.80 cm, the second component
travelled 2.50cm, and the third component
travelled 2.05 cm.

Plant Used: Malunggay Leaves (Moringa

Olifera)
Solvent-System used: Hexane-Acetone
Column Chromatography
In the column chromatography, three
eluates were produced mainly yellow green,
blue green, and green.

Color of Component

Volume of
eluate
(drops)

Yellow Green

60 drops

Blue Green

52 drops

Green

40 drops

Table 1. Color of components produced and

their respective volumes
Table 1 shows how many eluates were
produces and their respective volumes. The
first eluate, yellow green, yielded 60 drops.
The second eluate, blue green, yielded 52
drops and the last eluate, green, yielded 40
drops.
Figure 1. TLC plate after introducing to the
developing chamber.
The yellow green component contains
xanthophyll, which is a non-polar compound.
This can be confirmed by the column
chromatography. It was the first eluate to
come out of the column after using hexaneacetone, a non-polar compound. It follows
the concept of like dissolves like. Both
chlorophyll a and chlorophyll b contain
functional groups that make them polar.
Thus they dissolved more when acetone is
applied to the column.
The yellow component travelled the
farthest in the TLC plate due to it being a
non-polar compound. Polar compounds stick
more to the TLC plate as to compared to
non-polar compounds. As time passed, the
yellow component eventually fades, keeping
only the green components.

In Thin-Layer Chromatography, the Rf

values of the crude extract and the eluates
from column chromatography was computed
based on the distance travelled by the spot
divided by the spot travelled by the solvent
front. Since the Rf is a ratio, it has no unit of
measurement.
The Thin-Layer Chromatography produced
three different colors from the crude extract
and the eluates from Column
Chromatography. These were yellow green,
blue green, and green.
Color of the
Component

Yellow Green

Distance
of
compone
nt from
origin
(cm)

Rf Value

6.80cm

0.97

[2] Introduction to Chromatography

http://www.rpi.edu/dept/chem-eng/BiotechEnviron/CHROMO/chromintro.html

Computation of Rf values:

[3] Column Chromatography

http://orgchem.colorado.edu/Technique/Proce
dures/Columnchrom/Columnchrom.html
[4] Thin Layer Chromatography
http://orgchem.colorado.edu/Technique/Proce
dures/TLC/TLC.html
[5] Rf Values
http://www.marz-kreations.com/Chemistry/
Chromatography/Dyes/RF-Values.html
http://pharmainfo.net/msandhyasravya/blog
/column-chromatography
http://chemistry.csudh.edu/faculty/noel/CHE
317L/Thin%20Layer%20Chromatorgaphy
%20Experiment.htm
http://teaching.shu.ac.uk/hwb/chemistry/tut
orials/chrom/chrom1.htm

The computed values signify the ratio

of the distance travelled by the center of a
spot to the distance travelled by the solvent
front. In generally, it is the fraction of an
analyte in the mobile phase of a
chromatographic system.

REFERENCES
[1] Chromatography
http://www.discoveriesinmedicine.com/BarCod/Chromatography.html