EXPERIMENT 5

COLUMN AND THIN LAYER CHROMATOGRAPHY

Michael Edward D. Real, Kriselle Dianne M. Rosario,

James Urbano M. Santos, Angelyn M. Si, and Nicole Therese L. Siao
Group 9 2E MedTech Organic Chemistry Laboratory

ABSTRACT
Chromatography is a set of laboratory techniques for the separation of mixtures which involves passing a mixture dissolved
in a mobile phase through a stationary phase.This experiment aims to separate the colored components of red Siling Labuyo
and Malunggay leaves using column chromatography; to determine the purity of the components using thin layer
chromatography; and measure the Rf values of the colored components in thin layer chromatography. Column
Chromatography and Thin Layer Chromatography was used to separate the different colored pigments in Malunnggay leaves
and these pigments found to be Xanthophylls, the yellow eluate; Chlorophyll A, the dark green eluate; and Chlorophyll B,
the light green eluate . Colored eluate was collected through Column Chromatography and the purity of these eluates was
tested through Thin Layer Chromatography. UV lamp was used as the visualization method for the determination of
colorless substrates and the Retention or Retardation Factor was measured.

INTRODUCTION The silica gel got up to the intended part acted as

Column chromatography is the most general
method for the purification of organic compounds. It
involves the separation of individual compounds
from a mixture by passing it through a column
packed with silica gel. After the mixture is applied to
the column, an appropriate solvent is passed
through the column. Silica gel was used because
compounds will usually adhere to the silica to
different extents, they travel at different speeds
down the column. Each chromatographic zone
represents a natural constituent of the organism
from which the mixture was extracted. Thin layer
chromatography is very useful for rapid analysis of
organic mixtures. Plastic, metal, or glass plates are
prepared with a thin layer of silica gel. The organic
compound that was noticeable was the chlorophyll.
These are the green, magnesium containing,
tetrapyrrolic pigments of autotrophic plants. They
are easily convertible to other greens, yellow-green,
or gray pigments that are readily separable by
conventional chromatographic methods. Through
these processes the following objectives were met,
mainly:
1. Separate the colored components of siling
labuyo and malunggay leaves using column
chromatography.
2. Determine the purity of the components
using thin layer chromatography (TLC).
3. Measure the Rf values of the colored
components in TLC.
the stationary phase in the set-up.
Using a Pastuer pipette, 0.5 mL of the
extract was placed on top of the column. The
pigments were then eluted with the use of 10 mL of
hexane:acetone having a ratio of 7 is to3(7:3). The
column was properly observed upon every now and
then to avoid it to run dry. The solvent system was
then introduced into portions whilst discarding the
colorless eluate.
The collected colored eluates were placed in
separate test tubes. Note taking of the number of
drops of eluate that are collected in each tube
became an important task and was done cautiously.
Prepare a 5 x 8 TLC plate was prepared. With
this TLC plate, eluates were applied by spotting five
times. Spots were made sure to be a small one as
possible.Each spot were dried first before applying
the next eluate.
A developing chamber was then prepared by adding
10 mL of the hexane:acetone(7:3) solution and
lined the inner wall with filter paper, covered with a
watch glass and allowed to equilibrate.

EXPERIMENTAL
A. Compounds tested (or samples used)
10-15 leaves of malunggay, Hexane:Acetone(7:3
ratio), Acetone, Acetone Methanol.
B. Procedure
For the first procedure of this experiment,
10-15 malunggay leaves were selected and grinded
using a mortar and pestle. Using
hexane:acetone(7:3), pigments were extracted and
later on, a portion of the extracts were set aside for
TLC. After that, the column was plugged with cotton
at the bottom and uniformly packed with silica gel.
TLC plate was placed carefully in the
developing chamber to allow the solvent system to
rise up to 1 cm from the upper end before the plate
was removed from the chamber. Then, the solvent
front and air dry were marked immediately. After
many procedures had had done, components were
visualized using UV lamp in order to measure RF
values and to document the chromatoplate.
 In addition, to qualitative results, TLC can also
provide a chromatographic parameter known as Rf
Value. The Rf value is the retardation factor or the
ratio-to-front value expressed as a decimal fraction.
The Rf value is the ratio of the distance travelled by
the spot from the point of origin to the distance the
RESULTS AND DISCUSSION solvent travels. The general formula for computing
for the Rf value is
Table 1. Leaf Pigments and # of drops obtained to
get the pigment in Column Chromatography
# of drops
obtained Computations:
Leaf
Color before Chlorophyll A:
Pigment
getting the
pigment
Chlorophyll A Dark Green 75th drop
Chlorophyll B Yellowish Green 121st drop
Xantophyll Yellow 135th drop

The mobile phase slowly flows down through the Chlorophyll B:

silica gel column by gravity leaving behind zones of
color - the chromatogram. The theory of column
chromatography is analogous to that of thin-layer
chromatography. The different components in the
sample mixture pass through the column at
different rates due to differences in their partioning
behaviour between the mobile liquid phase and the Xantopohyll:
stationary phase.

The Rf values obtained will help us determine the

identity of a substance. If two substances on the
same TLC plate give spots in identical location, they
may be the same. But still, comparisons of Rf values
to be valid, TLC plates must be run under the exact
same conditions for stationary phase, mobile phase,
and temperature.

REFERENCE/S:

Books:
Pavia, D., Lampman, G., Kriz, G., & Engel, R.
(1998). Introduction to organic
laboratory techniques: a microscale
approach.3rd Ed. USA: Saunders Publishing
Comapany.
Figure 1. TLC plate
Pigments on the TLC plate after placing it on a Williamson, K; Minard, R; Masters K. (2007).
solvent system Macroscale and microscale organic
experiments. 5th Ed. Boston, USA: Houghton
Table 2. Thin Layer Chromatography: Leaf Mifflin Company.
Pigments and Distance travelled by the spotted
pigment from the origin Garner, CM. (1997). Techniques & experiments for
advanced organic laboratory. USA: John
Leaf Distance
Color Wiley & Sons. Inc.
Pigment (cm) MACAULAY, DB. (2007). General, organic &
Chlorophyll A Dark Green 6.0 cm biological chemistry an integrated approach.
Chlorophyll B Yellowish Green 5.9 cm USA: John Wiley & Sons. Inc.
Xanthophyll Yellow 5.0 cm

The distance travelled by the solvent of the

“solvent front”. In the experiment, we obtained 7.5
cm as the solvent front.