University of Santo Tomas

Faculty of Pharmacy
Organic and Inorganic Chemistry Laboratory

COLUMN AND THIN LAYER CHROMATOGRAPHY

OF MALUNGGAY LEAVES (Moringa Oleifera)

Janssen Nichole Q. Raca, ​Mark Aurelius C. Razon, Daniel Carlos S. Salipsip,John Michael L. Samiley, Mark M.
Serrano
Group 7 1H Medical Technology Inorganic and Organic Chemistry Laboratory

ABSTRACT

Chromatography is the separation of mixture used for the isolation of various substances. There are two of types and techniques
of chromatography used in this experiment. Column and Thin Layer Chromatography was performed in this experiment.
Malunggay leaves were used as the sample and its pigments were extracted using Hexane:Acetone (7:3). The process of Column
Chromatography, the extract was introduced in the column and eluate was collected in accordance with the eluents used in the
experiment where in the first eluent extracts yellow colored component which had 57 drops then the second eluent extracts dark
green colored component which had 50 drops and the third eluent extracts yellow-green colored component which had 44 drops .
While in the Thin Layer Chromatography, the purity of the components was determined and the Rf values were also calculated
on each pigment which also helped identify the polarity of each eluents used given that each has different Rf values where in the
eluent Hexane:Acetone had an Rf value of 0.79, Acetone had 0.77 Rf and Acetone: Methanol had 0.40 Rf. After finding out the
Rf value, Acetone: Methanol was the most polar while the least polar was Hexane:Acetone for the higher the Rf Value, the lower
the polarity it has and the lower the Rf value, the higher the polarity it has.

In Column chromatography the stationary phase, a

INTRODUCTION
solid adsorbent, is placed in a vertical glass (usually)
column. The mobile phase, a liquid, is added to the
Chromatography is a tool for the examination and
top and flows down through the column by either
separation of mixtures of chemical substances.It
gravity or external pressure. Column chromatography
involves the passage of a mobile phase which is a
is generally used as a purification technique: it
liquid solvent or mixture or solvent that use to carry
isolates desired compounds from a mixture. In Thin
the sample solutes under analysis along the paper and
Layer Chromatography, a liquid solution is directly
a across a stationary phase in a column which is the
applied to a solid adsorbent. Capillary action draws a
adsorbent, to separate the components in the mixture
developing solvent up the TLC plate. As this solvent
[1].Chromatography is an special separation process
passes through the spot, the mixture will be dissolved
given the following reasons: (a) separates complex
and will begin to move with the solvent front.
mixtures with great precision; (b) can purify basically
However, the adsorbent will also reabsorb part or all
any soluble or volatile substance if the right
of the mixture. As more solvent comes by, the
adsorbent material, carrier fluid, and operating
mixture will again go into solution, move further and
conditions are employed; (c) separates delicate
be reabsorbed. Since different materials will be
products since the conditions under which it is
dissolved and re absorbed at different rates,
performed are not typically severe; and (d) used to
separation will take place. The slide is removed from
separate the colored pigments in plants[2]. There are
the chamber once the solvent front reaches a
different techniques of chromatography where the
predetermined spot near the edge farthest from the
experiment focuses.This techniques includes Column
point of spotting. This passage of the solvent front
Chromatography and Thin Layer Chromatography.
through the adsorbent is known as developing the
plate. The extent of separation, measured by retention
factor or R​f​ value differences[3].
In chromatography, the retardation factor (R) is the
fraction of an analyte in the mobile phase of a
chromatographic system making the technique more
scientific rather than interpretation by sight. In planar
chromatography in particular, the retardation factor
Rf is defined as the ratio of the distance traveled by
the center of a spot to the distance traveled by the
solvent front.Identifying Rf Value is another way to
identify the Polarity of the solvents used[4].
The Objectives needed to be attained by the members
in this experiment are the ff: (1) to be able to separate
the colored components of malunggay leaves using
column chromatography, and (2) to measure the Rf
values of the colored components in TLC.
EXPERIMENTAL
A. Test Compound/s (or Sample/s) used
The sample used in conducting the experiment is the
malunggay leaves specifically 10 pieces of
malunggay leaves and the reagents and eluents used
were hexane:acetone (7:3) including acetone and
acetone:methanol (1:1).
B. Procedure
To start off with the experiment, 10 pieces of
malunggay leaves were placed in the pestle then
hexane acetone (7:3) was added and the leaves were
triturated using the mortar to extract its components
Figure 1. Trituration of the leaves
In Column Chromatography, in preparation of the
column,The Pasteur pipette was plugged with a small
piece of cotton and uniformly pack with silica gel up
to the intended part of the pipette then the column
was placed in an upright position on the iron sand and
secured using the clamp.The hexane acetone (7:3)
was added to the column until the silica gel was
completely wet.It is important to ensure that the
column don’t run dry.In separate test tubes, 5.0ml of
each eluents which are hexane acetone (7:3),acetone,
and acetone:methanol (1:1) were prepared.Then 10
drops of the extract of malunggay were added on the
top of the column using pasteur pipette.
Figure 2. Column Preparation
As the extract entered the column, 3 mL of each of
eluents were added in chronological order as listed
(hexane acetone (7:3),acetone, and acetone:methanol)
then the colored equated were collected in separate
vials and has its definite number of drops.to the
intended part of the pipette then the column was
placed in an upright position on the iron sand and
secured using the clamp.The hexane acetone (7:3)
was added to the column until the silica gel was
completely wet. It is important to ensure that the
column don’t run dry.In separate test tubes, 5.0ml of Figure 4. Preparation of the solvent used in TLC
each eluents which are hexane acetone (7:3),acetone,
and acetone:methanol (1:1) were prepared.Then the
extract of malunggay were dropped 10 times on the
top of the column using pasteur pipette.As the extract
entered the column, 3 mL of each of eluents were
dropped in chronological order as listed (hexane
acetone (7:3),acetone, and acetone:methanol) then the
colored equated were collected in separate vials and
has its definite number of drops.

Figure 3. Result of extraction from column

chromatography

In Thin Layer Chromatography, A 5 cm x 8 cm

predated TLC plate was prepared.One cm line from Figure 5. Spotting on the TLC plate
the bottom and the top of the plate were drawn. Then
the malunggay extract and the eluents were spotted
on the plate using capillary tubes with equal spaces in
order to avoid overlapping of the solutes.The
developing chamber was prepared by lining the inner
wall of the chamber with filter paper then placing
appropriate amount of the solvent
system:hexane:acetone (7:3) in the developing
chamber.The TLC plate is placed inside and covered
with a watch glass and was allowed to equilibrate
then the rising up of the solvent from the origin to the
solvent front was observed. After reaching the
solvent front,The plate was removed and lets it
dry.Visualized the components using the UV lamp
then the spots observed under the UV light were
encircled and measure to identify the Rf Value which
has a formula of Distance of solute/Distance of the
solvent in cm.
Figure 6. TLC chamber

After performing the TLC, The displacement of the

solute was measured by cm using a ruler then it will
used to get the Rf value. To get the Rf value of each
sample or eluents, this formula was used:
 The given figure shows the movement and
displacement of the solute from the point of origin up
to the solvent front in centimeters. Each were labelled
in accordance with its appropriate sample/eluent
name and then color of the eluate in accordance with
RESULTS AND DISCUSSION the result in the column chromatography. The Black
represents the displacement of the solvent which is
The results obtained from the experimentation are 4.7 cm.
shown as follows:
There are different colors that were extracted in
Table 1. Column Chromatography Results malunggay and each colored pigment were identified
as beta-Carotene (yellow), pheophytin a (green-gray)
and b (olive-green), chlorophyll a(blue-green) and b
Sample/Eluent Color of the Number
(yellow-green) and lutein (yellow) . Listed below are
s Sample and of Drops
the molecular structure of each pigment arranged
Eluates
from least polar to most polar:
Malunggay Green n/a

Hexane: Light Yellow 57

Acetone (7:3)

Acetone Dark Green 50

Acetone: Light green 44

Methanol (1:1)

The table above shows the result in column

chromatography where in there are different colors
produced with the different eluents along with the
extracted malunggay leaves.Hexane:Acetone
produces light yellow color,Acetone produces Dark
green color and Acetone:Methanol produces light
green color.It is visible that drops differ in number.In
that case, The least number of drops was the most
polar fluent which is Acetone:Methanol for it adheres
most to the layer of silica gels present in the Column
chromatography and silica gels are very polar in
nature that is why polar eluents descends slowly that
those who are least polar.

Figure 7.​ ​Thin Layer Chromatography results


The least polar pigment is the b-carotene which is ​a
hydrocarbon, making it quite non-polar and then
chlorophyll contains very ​polar bonds to magnesium
as well as a few ​polar functional groups.Then the
pheophytin are identical to chlorophyll a and b,
respectively, except that in each case the magnesium
ion Mg2+ has been replaced by two hydrogen ions
2H+.While Lutein and zeaxanthin is the most polar
for they belong to the class of carotenoids known as
xanthophylls and both contain hydroxyl groups. This
makes them more polar than carotenoids, such as
beta-carotene and lycopene, which do not contain
oxygen.
Table 2. Displacement of the solute and its Rf
values
Sample/Eluents Displacement Rf Value
of solute in cm
Malunggay 3.9 n/a
Hexane: 3.7 0.79
Acetone (7:3)
Acetone 3.6 0.77
Acetone: 1.9 0.40
Methanol (1:1)
The table above shows the displacement of each
solute measured in cm given that the TLC plate
has a total length of 8 cm and 2cm out of 8 cm
were the solvent front and the point of origin. The
Displacement of the malunggay is 3.9
cm,Hexane:Acetone is 3.7 cm, Acetone is 3.6 cm
and Acetone:Methanol is 1.9 cm.
Each displacement of solutes were divided to the
measure displacement of the solvent used which
is Hexane:Acetone and has a displacement of
4.7cm. The Eluents that has the highest Rf value
was the Hexane:Acetone while the lowest was
the Acetone:Methanol which means that the
Acetone:Methanol is the most polar eluents for
the higher the Rf value the lower the Polarity and
vice versa.That is why the displacement of the
Acetone:Methanol Solute has the least measured
displacement or ascends slower because the TLC
plate is made up of silica covered with
aluminum.Silica is a very polar substance.The
polar component will adhere to the silica tightly
and travel slowly up the plate while the least polar
or less polar will not adhere that strongly to the
silica and travel up relatively fast with the
solvent.The components that adhere more
strongly to the stationary phase travel more
slowly compared to those with weaker adhesion.
​Figure 8. Result of the TLC under UV light
CONCLUSION
Polarity of the sample and its rate of movement
are inversely related. The more polar spot travels
slower, and the less polar spot travels faster. Rf
values, on the other hand, are directly related to
the rate of movement. The fastest moving spot
has the highest Rf value.
RECOMMENDATION
In performing the experiment there are certain
things to consider to get the appropriate
result.First off, make sure that there are no
over-sized spots in TLC plate to avoid
overlapping and avoid uneven advance of solvent
front where in there should be a flat
 bottom,enough solvent and TLC plate must be cut
evenly.

REFERENCE

[1] Organic and Inorganic Chemistry Lab Manual

[2] (n.d.). Retrieved October 30, 18, from

https://www.chemguide.co.uk/analysis/chromatograp
hy/column.html

[3](n.d.). Retrieved October 29, 18, from

https://www.chem.ucla.edu/~bacher/General/30BL/ti
ps/TLC1.html
and so on

[4]​(n.d.). Retrieved October 31, 2018, from

http://dept.harpercollege.edu/chemistry/chm/100/dgo
dambe/thedisk/chrom/wback3.htm