THIN LAYER CHROMATOGRAPHY

Raphael Joseph Ledesma, Mico Jullano Lorenzo, Nathaniel Rhyan Lu, Nicole Anne Lubis, Jannina Luciano, Amiel Lance
Eric Xavier Magat
Group 5, 2C-Medical Technology, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Chromatography is a technique for separating and analyzing mixtures of chemicals by subjecting a mixture to various
processes. In this experiment two types of chromatography was used, both of which are under solid-liquid
chromatography. Different pigments of the siling labuyo were extracted with the use of DCM/C6H14 as eluant. In column
chromatography, the extract was introduced into the column and eluates were collected. Eluates were then subjected for
thin layer chromatography using a TLC plate. A UV lamp was then used to visualize the developed TLC plate. The
distance of the pigments was measured and retardation/retention factor (Rf) values were determined.

INTRODUCTION
Chromatography is a technique for
separating the components of a mixture on the
basis of the relative amounts of each solute
distributed between a moving fluid stream,
called the mobile phase, and a contiguous
stationary phase through which the mobile
phase passes and their affinities to both. The
mobile phase may be either a liquid or a gas,
while the stationary phase is either a solid or a
liquid (Giddings & Keller, n.d.). Analytes that
interact weakly with the stationary phase will
spend most of its time in the mobile phase and
move rapidly through the chromatographic
system. Analytes that interact strongly with the
stationary phase, on the other hand, will move
slowly. Gradually, as the analytes progress
through the system, they separate from each
other and can be found in their relatively pure
form
as
they
elute.
All
forms
of
chromatography work on the same principle
(Marsella, 2014).
It is a versatile process that can
separate very complex mixtures even in the
absence of previous detailed knowledge of the
number, nature or relative amounts of the
individual substances present.
Various types of chromatography are
possible, depending on which two phases are
used. These are solid-liquid (column, thin
layer), liquid-liquid (paper, high-performance

liquid),
and
gas-liquid
chromatographic methods.

(vapor-phase)

Thin-layer chromatography (TLC) is a

chromatographic technique that is often used
to monitor the progress of organic reactions
and to check for the purity of the components.
Thin-layer chromatography consists of a
stationary phase immobilized on a glass or
plastic plate, and an organic solvent. The
sample, either liquid or dissolved in a volatile
solvent, is deposited as a spot on the
stationary phase. The constituents of a sample
can be identified by simultaneously running
standards with the unknown. TLC can also be
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.
The retention/retardation factor, or Rf, is
defined as the distance traveled by the
compound divided by the distance traveled by
the solvent.
The main objectives of the experiment
were: (a) to determine the purity of the color
components in siling labuyo collected through
column chromatography, by the use of TLC
and, (b) to measure the retention or retardation
factor (Rf) values of the colored components in
TLC.
MATERIALS AND METHOD

I.

MATERIALS
The materials used for this experiment
are: DCM/C6H14 (Dichloromethane hexane),
a beaker, a watch glass, filter paper, cotton,
capillary tubes, test tubes, a ruler, a TLC
plate and a UV lamp.

II.

METHOD

First, 1.0 cm was measured on both the

upper and lower sides of the 5 cm x 8 cm
precoated TLC plates to allow for more
accurate marking of the solvent front and
origin.
Next, the collected colored eluates were
applied evenly on the origin of the TLC
plate, by spotting 10 times, as small as
possible, using capillary tubes. Each spot
was allowed to dry first before the next was
applied.
A developing chamber was then
prepared using a beaker filled to a depth of
just less than 0.5 cm with DCM-hexane
(1:1), which will be used as the solvent
system. The inner wall of the chamber was
lined with filter paper, the top was covered
with a watch glass putting cotton at the
spout of the chamber, and was allowed to
equilibrate.
The TLC plate was then placed carefully
in the developing chamber and the solvent
system was allowed to rise up to 1 cm from
the upper end. After which, the plate was
carefully removed from the chamber. The
plate was air-dried.
After drying, components were then
visualized using UV lamp, to allow for a
clearer viewing. The colored spots were
then marked and measured. Rf values were
calculated and the chromatoplates were

documented. Rf values were calculated

using the formula below:

RESULTS AND DISCUSSIONS

Three eluates were produced from the
crude siling labuyo extract. Each had different
colors/pigments as shown in the table below.
The table also shows the distance travelled by
each. The crude extract was also included for
reference.
Table 1. Thin Layer Chromatography: Colors and distance
travelled by the colored spots from the origin

Color

Distance (cm)

Dark orange (Crude)

2.1 cm

Yellow

0.0 cm

Tomato red

0.0 cm

Light orange

0.0 cm

As shown in the table, only the crude

extract travelled across the TLC plate. It moved
2.1 cm. No other spots were found.
The corresponding Rf value for the
crude extract is 0.42, having no unit as it is a
ratio of two same measurements.

A UV lamp was used to visualize the

colorless compounds. The TLC plate, which is
the stationary phase, is made up of substances
that fluoresces in UV light. A plate placed
under a UV lamp will glow, except for spots
which are the organic compounds. Only one
spot appeared under the UV lamp, and this
was crude extract.
There are a number of reasons why there
were no results gathered in thin layer
chromatography for the other components.
First, of course we cannot rule out human
error. Another could be the lack of enough

compound spotted, or perhaps because the

solution of the compound was too dilute.
Lastly, the eluents could have evaporated
while they were in storage, as the solvent
systems used to collect them are very volatile
compounds.
REFERENCES
Giddings, J. C., & Keller, R. A. (n.d.). chromatography
(chemistry) -- Encyclopedia Britannica. Retrieved September
21,
2014
from
http://www.britannica.com/EBchecked/topic/115917/chromatog
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"Chromatography." (n.d.). Chemicool Periodic Table. Retrieved
September
21,
2014
from
http://www.chemicool.com/definition/chromatography.html
Marsella, G. (2014). What is chromatography - definition, types
& uses. Retrieved September 21, 2014 from http://educationportal.com/academy/lesson/what-is-chromatography-definitiontypes-uses.html
"Thin-layer chromatography." (n.d.). Chemicool Periodic Table.
Retrieved
September
21,
2014
from
http://www.chemicool.com/definition/thin_layer_chromatograph
y_tlc.html
University of Colorado-Boulder, Department of Chemistry and
Biochemistry. (2014). Thin Layer Chromatography (TLC).
Retrieved
September
21,
2014
from
http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.h
tml