Extraction of colored pigments present in malunggay leaves using column and thin layer

chromatography
*Rifareal,Katrina Isabel C., Rodriguez, Alecs Dale M., Pornillos, Ma. Eloisa
Joanne E., Quezada, Ester Florentina W., Rapinan, Erika Faye D., Rariza, Mary Chelsea
S.
Abstract
Chromatography is a physical method of separation in which the components to
be separated are distributed between two phases one of which is stationary
(stationary phase) while the other (mobile phase) moves in a definite direction. Of all
the various types of chromatography, the one used in this experiment was Column and
Thin Layer Chromatography. Column Chromatography is a separation technique in
which the stationary bed is within a tube, while Thin Layer Chromatography involves a
stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a
flat, inert substrate. In this experiment, the colored pigments of malunggay leaves were
extracted using hexane-acetone (7:3). After the obtainment of the extract, it was then
separated using Column Chromatography. The resulting eluates were applied on a TLC
plate to undergo Thin Layer Chromatography which aids in determining the RF values
of the components. Four colored components of the malunggay leaves were observed
in the outcome of the experiment, and the coloreless substrates were identified with the
use of a UV lamp.
Introduction:
Chromatography is an analytical
technique used for separating a mixture
of chemical substances into its
components so that these components
can be identified or analyzed. It is done
by the distribution between two phases,
mainly the stationary phase and the
mobile phase. The major underlying
principle in chromatography is that the
stationary phase will always be more
polar than of the mobile phase meaning,
the separation of components will be
achieved if one component adheres to
the stationary phase than the mobile
phase. Components that interact more
with stationary phase will move slowly
thus resulting to different partition
coefficients and rate between stationary
phase and mobile phase.
There are different types of
chromatography depending on the two
phases used. One of these phases is the
solid-liquid wherein column and thin
layer chromatography are used. Another
type is the liquid-liquid which uses paper
and high performance liquid
chromatography; and the gas-liquid
which uses vapour-phase
chromatography.
In the experiment, Column and
thin layer chromatography were used.
The objectives of the experiment is to
identify the colored pigments or
components present in malunggay
leaves, determine the purity of the
components using thin layer
chromatography and calculate the Rf
values of the components in thin layer
chromatography.
Column chromatography is the
selective adsorption and separation of
mixture through which a suitable solvent
has passed. It is used to determine the
number of components in a mixture and
it separates as well as purify the
quantities of components for analysis.
Thus this method is very time
consuming and needs technical skill.
On the other hand, thin layer
chromatography is used to separate
non-volatile mixtures. It can easily
determine the number of components of
a mixture sample. It even allows the
identification of components. It requires
relatively small amount of sample thus
lessening the expense. However, the
spotting of the samples is often over
large and there is the presence of
uneven advance of solvent front
resulting to incomplete separation of the
components.
The Retention factor is the ratio
of the distance travelled by the
component and the distance travelled by
the solvent, in this experiment is the
hexane-acetone (7:3).
Rf=Distance travelled by the component
Distance travelled by the solvent



Methodology:
Pigments of malunggay leaves
were extracted using hexane-acetone
with the ratio of 7:3. Some portion of
the extract was set aside for thin layer
chromatography (TLC).
The column chromatography was
set up by plugging cotton at the bottom
of the pipette then followed by a
uniformly packed silica gel up to the
intended part.
0.5 mL of the extract was placed
on top of the column and 5.0 mL of
hexane-acetone with the ratio of 7:3,
acetone and acetone-methanol with the
ratio of 1:1 were introduced in
succession not allowing the column to
run dry. The collected colorless eluate
was discarded while the colored eluates
were collected in different test tubes as
the color changes. The number of drops
of collected eluates was noted.
After collecting the eluates from
column chromatography, TLC was
performed.
The eluates were applied on a
5cm x 8cm percoated TLC plate by
spotting 10 times. Each spot was
allowed to dry first before applying
another one. The spots were made sure
that they are small enough to prevent
from disarrangement of the colors.
The developing chamber was set
up by placing a sufficient amount of
hexane-acetone with the ratio of 7:3 in
a beaker. The inner wall of the beaker
was lined with a filter paper and the
beaker was covered with a watch glass
allowing the developing chamber to
equilibrate.
The plate was placed carefully in
the developing chamber allowing the
solvent system to rise up 1 cm from the
upper end of the plate. Then the plate
was carefully removed from the
developing chamber and the solvent
front was marked. Then the plate was
air dried.
The components were visualized
by using a UV lamp. The Rf values were
calculated and the chromatoplates were
documented.
Results:
Plant used: Malunggay leaves
Solvent System used: Hexane-acetone
(7:3)
Column Chromatography
Color of
Component
Volume of
Eluate
(Drops)
1 Green 15 drops
2 Dark Green 105 drops
3 Light Yellow 70 drops
4 Colorless 75 drops
Table 1 Column Chromatography (Table
of Results)
Thin Layer Chromatography
Color of
Component
Distance of
Components
from origin
(x) in cm
Rf
Value
1 Green a. 2.50 cm
b. 3.00 cm
0.338
0.405
2 Dark Green a. 0.70 cm
b. 1.30 cm
c. 1.90 cm
d. 2.50 cm
e. 3.00 cm
0.095
0.176
0.257
0.338
0.405
3 Light
Yellow
a. 1.30 cm
b. 1.90 cm
0.176
0.257
4 Colorless a. 0.70 cm
b. 1.90 cm
c. 2.50 cm
0.095
0.257
0.338
Table 2 Thin Layer Chromatography
(Table of Results)








Figure 1 Developed TLC Plate (Viewed
under Ultraviolet Lamp)
Computations for Rf (Retention Factor):
Rf=Distance travelled by the component
Distance travelled by the solvent
Distance travelled by solvent: 7.40 cm
1. Green
a. 2.50 cm/ 7.40 cm = 0.338
b. 3.00 cm/ 7.40 cm = 0.405
2. Dark Green
a. 0.70 cm/ 7.40 cm = 0.095
b. 1.30 cm/ 7.40 cm = 0.176
c. 1.90 cm/ 7.40 cm = 0.257
d. 2.50 cm/ 7.40 cm = 0.338
e. 3.00 cm/ 7.40 cm = 0.405
3. Light Yellow
a. 1.30 cm/ 7.40 cm = 0.176
b. 1.90 cm/ 7.40 cm = 0.257
4. Colorless
Solvent
front

Origin

a. 0.70 cm/ 7.40 cm = 0.095
b. 1.90 cm/ 7.40 cm = 0.257
c. 2.50 cm/ 7.40 cm = 0.338
Discussion:
In reference to Table 1, three
eluates were produced from the
extraction of colored pigments in
malunggay leaves using the column
chromatography. The colors produced
were green, dark green and light yellow.
The volume of green is 15 drops; dark
green is 105 drops and light yellow is 70
drops.
In reference to Table 2, there
were only 3 visible spots on the TLC
plate: green, light green and pink.
These are all from the second eluate
(left to right) extracted from column
chromatography. The rest of the colors
which cannot be seen by the naked eye
were viewed under the UV lamp.
After measuring the distance
travelled by the components from the
origin, the Rf value or the retention
factor of each component is calculated.
The retention factor is defined as the
distance travelled by the component
divided by the distance travelled by the
solvent. For the first eluate (left to
right), the Rf value of the first
component is 0.338 and the second
component is 0.405. For the second
eluate the Rf values are 0.095, 0.176,
0.257, 0.338 and 0.405 respectively. For
the third eluate the Rf values are 0.176
for the first component and 0.257 for
the second component. Lastly for the
last eluate, the Rf values are 0.095,
0.257 and 0.338 respectively.
The components will differ in
solubility and in strength of their
adsorption to the adsorbent which
results to different distances of the
components as well as the Rf values.
The Rf values of the components are
constant given that the solvent system,
adsorbent and amount of eluates
spotted are also constant. The larger
the Rf value of the component, the
larger the distance it travels on the TLC
plate. The Rf, however, can confirm the
identity of the component. If the
identities of the components are still
unknown, the spots on the TLC plate
can be the basis. If two components
have the same Rf value, they likely to
have the same identity therefore they
likely to be the same compound. Hence,
if they have different Rf values then
they are different compounds having
different identities.
The developed plate wasnt able
to show the complete separation of the
colored pigments because of the errors
in spotting. One possible cause is that
the spots were not completely dried
before applying another spot. The spots
were also not small enough that caused
disarrangement of the colors. Lastly, the
developing chamber was not
immediately covered by the watch glass
resulting to lack of development of the
plate.
References:
Thin Layer Chromatography (TLC).
UCB. 12 August 2014.
http://orgchem.colorado.edu/Technique
/Procedures/TLC/TLC.html
Ruppel Jr.,I.B., et al. (1971). Column
and thin-layer chromatography. Journal
of Chemical Education. 48(9) p. 635.
DOI: 10.1021/ed048p635
Chromatography. 18 September 2014.
http://iupac.org.html
General Introduction to
Chromatography, Various Techniques
and Its Application in Pharmaceutical
and Chemical Industries.
http://shodhganga.inflibnet.ac.in.html
Thin Layer Chromatography. 2007.
http://www.chemguide.co.uk/analysis/c
hromatography/thinlayer.html