COLUMN AND THIN LAYER CHROMATOGRAPHY

Mark Paul P. Pastrana, Mariah Ericka M. Patawaran, Princess Juneire M. Peligro,

Francisco Q. Pua III, Rose Anne L. Quyo and Janille P. Ragpa
Group 8 2B Medical Technology Organic Chemistry Laboratory

ABSTRACT
The main objectives were to separate the colored components of malunggay leaves by means of column
chromatography, as well as to determine the purity of the components using thin layer chromatography
(TLC) and measure the Rf values of the colored components obtained herein. For column chromatography,
the sample prepared was loaded into a Pasteur pipette plugged with cotton and uniformly packed with
silica gel. The eluents used were 7 mL hexane:acetone (7:3), 5 mL hexane:acetone (1:1), 5 mL acetone,
and 5 mL acetone:MeOH (1:1); the collected eluates were dark green, light green, yellow green, and
yellow. For thin layer chromatography, the eluates were applied in spots on a TLC plate pre-coated with
silica and placed in a developing chamber lined with filter paper and hexane:acetone (7:3) as the solvent
system. The solvent front was measured to be 8.2 cm. The TLC plate was then put under a UV lamp and
the Rf values were computed as follows: Dark green0.5, Light Green0.49, Yellow Green0.46,
Yellow0.99, Pink0.32, and Bluish-violet0.20.

INTRODUCTION
Chromatography is a technique used to
separate mixtures into components as a result of
differential partitioning behavior between a
stationary phase and a mobile phase that
percolates through the stationary bed.
[1, 2]
Column and thin layer chromatography are
examples of the solid-liquid method of
chromatography, which utilizes an adsorbent
solid stationary phase (silica) and a liquid mobile
phase. In column chromatography, the solid
stationary phase is situated within a tube as the
liquid mobile phase is added to the top and
allowed to flow down through the tube
[3]
; in thin
layer chromatography, the stationary phase is
pre-coated on a plate. Column chromatography
employs both solubility and adsorptivity for the
stationary phase.
[4]
Thin layer chromatography is
usually used to determine the purity of the
components of a mixture, and is helpful in
determining the identity of an unknown
substance based on its Retention factor (Rf)
value.
[3]


The experiment aimed to: separate the
components of malunggay leaves using column
chromatography, determine the purity of the
components using thin layer chromatography
(TLC), and measure the Rf values of the colored
components in TLC.
[4]
EXPERIMENTAL
A. Compounds tested (Samples used)
Sample: Malunggay leaves

Malunggay (Moringa oleifara) is a plant
widely known for its nutritional value and herbal
uses. It is native to countries with a tropical
climate, such as the Philippines, Africa, and
India
[6]
,

as well as countries in Central and South
America. Almost all parts of the malunggay

tree
may be used for practical reasons, and requires
only minimal care for maintenance.
For the experiment, malunggay leaves
were used as a source for the colored
components in both methods of chromatography
employed.


Figure 1. Malunggay leaves



B. Procedure
1. Sample Preparation
Twenty-five pieces of malunggay leaves were
titurated using a mortar and pestle, and 5 mL of
hexane:acetone (7:3) was added to the sample.
2. Column Chromatography
The set-up for column chromatography was
prepared by positioning an iron clamp on the iron
stand. The column used was a Pasteur pipette
plugged with cotton which served as a bed
support for the stationary phase; silica gel was
then uniformly packed into the tube. The column
was then clamped onto the set-up. Eluents for
the procedure were obtained and placed in test
tubes: 7 mL hexane:acetone (7:3), 5 mL
hexane:acetone (1:1), 5 mL acetone, and 5 mL
acetone:MeOH (1:1). The sample was loaded into
the column, followed by the addition of
hexane:acetone (7:3) by using a dropper. The
colored eluates were collected into test tubes,
noting the number of drops per eluate, while
colorless eluates were discarded. Without letting
the column run dry, hexane:acetone (1:1) was
introduced into the column, and in the same
manner the eluates were collected. This was the
same for the succeeding eluents, and went on
until no more colored eluates could be obtained
from the column.

Figure 2 Column Chromatography
3. Thin Layer Chromatography
The eluates obtained from column
chromatography were applied on a TLC plate pre-
coated with silica by spotting it seven times per
color using a capillary tube. Each spot was dried
before applying the next.
A developing chamber was prepared by
placing an amount of the solvent system,
hexane:acetone (7:3), into a beaker. Filter paper
was used to line the walls of the beaker, and was
then covered with a watch glass to equilibrate the
chamber. Once the filter paper was saturated
with the solvent system, the TLC plate was
carefully placed in the beaker to develop.
When the solvent system had reached about
a centimeter from the upper end of the TLC
plate, the plate was removed, and before
allowing it to air-dry, the solvent front was
marked. Once air-dried, the plate was placed
under a UV lamp to visualize the components to
determine any additional colors that were
invisible without UV light.


Figure 3.1 Thin Layer Chromatography


Figure 3.2 Thin Layer Chromatography
RESULTS AND DISCUSSION
In column chromatography, four colored
components were obtained: Dark Green, Light
Green, Yellow Green, and Yellow. As seen in
Table 1, Light Green was the eluate collected the
most in terms of number of drops, while yellow
was the least collected.
Iron stand
Iron clamp
Pasteur
pipette
Silica gel
Cotton
Sample
Watch Glass
Beaker
TLC
plate
Solvent
system
Filter
paper
Color of Component Volume of eluate
(drops)
Dark Green 67
Light Green 200
Yellow Green 37
Yellow 14
Table 1 Column Chromatography results
In thin layer chromatography, the solvent
front was measured to be 8.2 cm. Two more
colored components were produced under the UV
lamp: pink and bluish-violet. The distances from
the origin of every colored component were
measured (in centimeters) and the component
that travelled the farthest was Yellow, while the
component that travelled the shortest was bluish-
violet. This can be seen in Table 2.
Color of Component Distance from the
origin (X) in cm
Dark Green 4.1 cm
Light Green 3.6 cm
Yellow Green 3.8 cm
Yellow 8.1cm
Pink 2.6 cm
Bluish-violet 1.6 cm
Table 2 Distance per Component from the origin
obtained from thin layer chromatography
The Retention factor (Rf) value was computed
for each component by obtaining the quotient of
the distance each component travelled from the
origin and the distance travelled by the solvent
(solvent front).
The Rf value can used to determine the
polarity of two different compounds; the one with
a larger Rf value is less polar because it has low
affinity to the polar adsorbent on the TLC plate.
The Rf value can also be used to determine the
identity of an unknown compound. If an unknown
compound is experimentally proven to have the
same Rf value with a known substance, then it is
likely that they are the same compound.
[5]

The mathematical equation for Rf value is as
follows:

Dividing each components distance from the
origin by the distance travelled by the solvent,
the following values were obtained:
Color of Component Rf value
Dark Green 0.5
Light Green 0.49
Yellow Green 0.46
Yellow 0.99
Pink 0.32
Bluish-violet 0.20
Table 3 Computed Rf values for each component
REFERENCES
[1] Brian M. Tissue. Chromatography.
http://www.files.chem.vt.edu/chem-
ed/sep/chromato.html. Retrieved 8/11/13
[2] Rebecca Carrier and Julie Bordonaro. Intro to
Biochemical Engineering
http://www.rpi.edu/dept/chem-eng/Biotech-
Environ/CHROMO/chromintro.html.Retrieved
8/11/13
[3] University of Colorado at Boulder,
Department of Chemistry and
Biochemistryhttp://orgchem.colorado.edu/Techni
que/Procedures/Columnchrom/Columnchrom.htm
l
[4] Bayquen, A., Cruz C., de Guia, R., Lampa, F.,
Pea, G., Sarile, A. and Torres, P. (2009).
Laboratory Manual in Organic Chemistry.
Manila: C & E Publishing, Inc.
[5] University of Colorado at Boulder. Thin Layer
Chromatographyhttp://www.ce.gxnu.edu.cn/orga
nic/net_course/content/tlc/Retention_Factor.htm

[6] Medical Health Guide. What is
Malunggay.http://www.medicalhealthguide.com/
articles/malunggay.htm