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Formal Report Expt 5
Formal Report Expt 5
Faculty of Pharmacy
Organic Chemistry Laboratory
Abstract
Chromatography is a way of separating and analyzing the constituents of a mixture by passing
them through an inert stationary environment using a mobile solvent that carries them. The
two different types of Chromatography used to achieve the objectives of the experiment were
the Column Chromatography, which is a preparative technique where the eluates were taken,
and the Thin Layer Chromatography, which is a fast technique that required only a small
amount of the material. The objectives of this experiment were to illustrate the principles of
chromatographic separations, and to demonstrate how Thin Layer Chromatography and
Column Chromatography are used in Organic Chemistry. In the experiment, the 3 eluates
collected from the extract of Malunggay leaves using the process of Column Chromatography
were the yellow-green colored component (1 st eluate), which had 60 drops, the blue-green
colored component (2nd eluate), which had 52 drops, and the green colored component (3 rd
eluate), which had 40 drops. After spotting these eluates on the Thin Layer Chromatography
plate and placing the plate in a developing chamber, it was evidently seen that from the origin,
the yellow-green, blue-green, and green eluates travelled a distance of 6.80 cm, 2.50 cm, and
2.05 cm, respectively. The UV lamp was then used to visualize the TLC plate and Rf values
were computed, giving the yellow-green eluate a 0.97 Rf value, the blue-green eluate a 0.36
Rf value, and the green eluate a 0.60 Rf value.
INTRODUCTION
Chromatography, first introduced by the Russian botanist Micharl Iswett, is a separation
method that relies on differences in partitioning behavior between a flowing mobile phase,
which is a liquid solvent (or mixture of solvents) that use to carry the sample solutes under
analysis along the paper, and a stationary phase, which is the adsorbent, to separate the
components in a mixture. [1] Chromatography is a very special separation process for a
multitude of reasons: (a) separates complex mixtures with great precision; (b) can purify
basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and
operating conditions are employed; (c) separates delicate products since the conditions under
which it is performed are
not typically severe; and (d) used to separate the colored pigments in plants. [2]
In Column Chromatography, the stationary phase, a solid adsorbent, is placed in a vertical
glass (usually) column. The mobile phase, a liquid, is added to the top and flows down through
the column by either gravity or external pressure. Column chromatography is generally used
as a purification technique: it isolates desired compounds from a mixture. [3]
Thin Layer Chromatography is a simple, quick, and inexpensive procedure that gives the
chemist a quick answer as to how many components are in a mixture. TLC is also 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 (preferably both run on the same TLC plate). A TLC plate is a
sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually
silica or alumina). [4]
In order to make the technique more scientific rather than a mere interpretation by sight,
Retention Value (Rf value) was applied in chromatography. A particular compound will travel
the same distance along the stationary phase by a specific solvent (or solvent mixture) given
that other experimental conditions are kept constant. In other words, every compound (dye,
pigment, organic substance etc.) have a specific Rf value for every specific solvent and solvent
concentration. Rf values come very handy for identification because one can compare Rf values
of the unknown sample (or its constituents) with Rf values of known compounds. [5]
In this experiment, the group should be able to attain the following objectives: (1) to be able
to separate the colored components of malunggay leaves using column chromatography, (2) to
determine the purity of the components using think layer chromatography (TLC), and (3) to
measure the Rf values of the colored components in TLC.
Methodology
Experimental:
Compounds tested (or Samples used)
Leaves of Malunggay (Moringa oleifera),
Hexane: Acetone (7:3), Acetone, Acetone
Methanol (1:1)
B.
Procedure:
1. Column Chromatography
Pigments of Moringa oleifera
were extracted by grinding the
leaves with a mortar and pestle
and adding hexane-acetone (7:3).
A portion of the extracts was then
Figure 4. Hexane-acetone,
acetone, and acetone-methanol
eluents
Figure 5. Developing Chamber
The plates were removed after
from the chamber and the solvent
front was immediately marked.
The plates then were air-dried.
After the results were obtained,
the components were visualized
through the use of UV lamp. The
data attained was used to
measure the distance traveled by
the spot and utilized for the
computation of Rf values.
Blue Green
2.50cm
0.36
Green
2.05cm
0.29
Color of Component
Volume of
eluate
(drops)
Yellow Green
60 drops
Blue Green
52 drops
Green
40 drops
Yellow Green
Distance
of
compone
nt from
origin
(cm)
Rf Value
6.80cm
0.97
Computation of Rf values:
REFERENCES
[1] Chromatography
http://www.discoveriesinmedicine.com/BarCod/Chromatography.html