THIN LAYER CHROMATOGRAPHY

Group 6

Pharmacy

Organic Chemistry Laboratory

ABSTRACT
Chromatography is a physical method that is used in the separation and analyzation of components in a mixture. The
experiment aimed to determine the purity and Retention factor value of the colored pigments through Thin Layer
Chromatography (TLC). In this experiment, pigments of malunggay were extracted and colored eluate was collected
through the Column Chromatography method. Afterwards, the purity of the components were determined through the
TLC method, and the developed TLC plate was used in order for the Retention Factor value to be measured.

INTRODUCTION
Chromatography is a technique that is
widely used for identifying and analyzing
mixtures through the separation of various
chemical
compounds.
Different
types
of
chromatography are very useful for analytical
purposes, depending on the physical state of the
phases used. In this experiment, Column
Chromatography was used to separate the
organic
compounds
of
malunggay.
The
components were distributed in a stationary and
mobile phase and were carried along to each
phase in varying degrees. The column was not
allowed to run dry as the eluates were collected.

plate which can be a strip or piece of glass,

metal, or rigid plastic. Small amounts of the
mixture to be analyzed were also spotted near
the bottom of the TLC plate. Through capillary
action, the solvent then travels up the plate.
Thin Layer Chromatography is a fast, efficient,
and a very simple method to use.
The aim of this experiment was to determine
the purity of the components of malunggay and
measure its Retention Factor value through the
use of thin layer chromatography.

EXPERIMENTAL
The compound tested was malunggay and
after it underwent through the process of column
chromatography, 4 eluates were yielded from the
extraction. Yellow, Green, Light Green, and Light
Yellow were extracted respectively; also, the
number of drops per color was noted.

Figure 1. Column Chromatography Set-up

Figure 2. The Eluates Obtained
Another method used in this experiment
that this paper will be focusing about is the Thin
Layer Chromatography. TLC is a qualitative,
analytic technique used to initially check on the
identity of a given sample. It is then used to
check the purity of a compound or determine the
number of components in a mixture or column
chromatograph
function.
The
Thin
Layer
Chromatography method is advantageous since it
is simple, quick, inexpensive, and requires only
small amounts of the sample. This technique is
also closely related to column chromatography.
The absorbent is coated on one side of a TLC

Each of the colored eluates were spotted 10

times on a pre-coated TLC plate through the
usage of a capillary tube. Each spot of the
eluates were made as small as possible, and
were dried before each application. The
developing chamber was prepared with a 7:3
hexane-acetone solvent, with its inner walls lined
with filter paper, it was then covered with a watch
glass, and was allowed to equilibrate. The TLC
plate was placed in the developing chamber as
the solvent system was allowed to rise up to 1cm
from the upper end. The TLC plate was removed

from the chamber as its solvent front was

immediately marked and was then allowed to airdry. Lastly, the plate was visualized using a UV
lamp and its RF values were computed.

the pigments found in malunggay leaves are as

follows: carotene, Xanthophylls, Chlorophyll A, and
Chlorophyll B. The Retention Factor value was
computed using the formula:
Rf= distance traveled by the compound/
distance traveled by the solvent

Figure 3. The Developing Chamber Set-up

Figure 4. TLC plate under UV lamp

RESULTS AND DISCUSSION

Plant used: Malunggay
Solvent system used: Hexane-acetone
Table 1. Thin Layer Chromatography
Color of
Component

RF Value

The
compounds
extracted
from
the
malunggay
leaves
were
colored,
thus,
its
visualization can be straightforward. UV lamps are
used to visualize the colorless compounds on the
plate. Also, the thin layer plate has a substance
added to it that will fluorescence when exposed to
UV light. Thus, making it glow under a UV lamp.
Such spots are invisible to the naked eye, and the
glow present is masked at the position where the
spots are at the final chromatogram.

Yellow

Distance of
component from
origin (X) in cm
4cm

Green

3.5cm

0.54

Light Green

2cm

0.30

REFERENCES

Light Yellow

0.5cm

0.07

[1] de Grano, R.V., Esmalla, M.S., Liu, M.I. and

Pena, G. (2016). General Chemistry
Laboratory Manual Revised ed. Diliman,
Quezon City.: MaxCor Publishing House Inc.
[2] Thin Layer Chromatography
http://chem.libretexts.org/Core/Analytical_Chemi
stry/Lab_Techniques/Thin_Layer_Chromatograph
y
[3] Using Fluorescence: TLC
http://www.chemguide.co.uk/analysis/chromatog
raphy/thinlayer.html

0.62

The results shown on table 2 appeared to be

consistent with the actual values of the said
components. The most polar is the light yellow
component which showed to have travelled the
slowest and resulted to have the lowest RF value.
Whereas, the most non-polar is the yellow
component. This is because it showed to have
travelled the farthest and even resulted to have the
highest RF value. According to additional research,