Biochromatography: Phytochemical Screening

(Villarta, Feldan P.)

Abstract: The experiment is aimed at screening the ethanolic extract of Euphorbia hirta for the
presence of alkaloids, phenols, flavonoids, saponins and tannins. Thin-layer chromatography was
used to analyze the secondary metabolites in the plant. The extract contains phenols, saponins
and flavonoids.
Introduction
Phytochemicals are a large group of plantderived compounds hypothesized to be
responsible for much of the disease
protection conferred from diets high in
fruits, vegetables, beans, cereals, and plantbased beverages such as tea and wine (Arts
and Hollman, 2005).
Phytochemicals have provided the basis for
numerous commercial medications used
today for the treatment of a wide range of
diseases such as high blood pressure, pain,
asthma, and cancers (Park, 2006). Thus,
whether a specific plant contains
phytochemicals needs to be investigated.
The experiment specifically aims to screen
the ethanolic extracts of Euphorbia hirta for
the presence of alkaloids, phenols,
flavonoids, saponins and tannins.
Experimental Methods
A. Materials, Chemicals and Apparatus
The following chemicals were used during
the experiment: 95% ethyl alcohol, silica gel,
chloroform, methyl alcohol, ferric chloride
solution, vanillin, sulfuric acid, hydrochloric
acid, formic acid, and distilled water.
The following apparatus were utilized during
course the experiment: capillary tube,
Erlenmeyer flask, rotary evaporator, TLC
plate, beaker, aspirator, pipet, sprayer,
spatula, graduated cylinder, analytical
balance, funnel, vial and grinder.

B. Collection of Plant Sample

Fresh plant samples of Euphorbia hirta were
collected at the vicinities of Central
Mindanao University. Only plant samples
showing no signs of infections were picked.
The samples were washed with tap water
and rinsed with distilled water. They were
dried in an oven at 40C.
C. Extraction
The dried plant samples were ground to
reduce the size. About 50 g of the ground
plant sample was weighed and treated with
sufficient 95% EtOH to completely submerge
the material in an Erlenmeyer flask. The flask
was stoppered and the material was kept
soaked for 24 hours.
The mixture was then filtered. The flask and
plant material were rinsed with fresh
portions of 95% EtOH. The washings and the
plant material were transferred into a
funnel. The plant residue was discarded. The
filtrate was then concentrated under vacuo
at temperatures not exceeding 40C to
dryness. The extract was then transferred in
a vial and stored in the refrigerator.
D. Preparation of TLC plates
Silica gel was used as the coating material. A
slurry of silica gel was prepared. The slides
were coated by dipping them onto the
container of slurry. The coated plates were
then placed in oven at 105C for drying
purposes.

E. Phytochemical Screening
The extract was screened for the presence of
alkaloids, phenols, flavonoids, saponins and
tannins.
Before each test, a dried coated plate was
spotted using a capillary tube. The capillary
tube was dipped in a container containing
the extract dissolved in ethanol. The spot
was about 1 cm from the bottom of each
plate.
Test for Alkaloids
The spotted plate was placed in a chamber
(beaker) containing chloroform and
methanol in a 5:1 ratio. After the solvent has
reached the top of the plate, it was removed
and air-dried. It was sprayed with
Dragendorffs reagent. The positive result
for this test is the formation of orange spots.
However, it was not seen during the
experiment implying a negative result as
shown in Figure 1. Yellow spots were seen.
Test for Phenols
The spotted plate was placed in a beaker
containing ethyl acetate, formic acid, acetic
acid and water in a 100:11:11:25 ratio
respectively. After the solvent has spread up
to the top of the plate, it was removed and
air-dried. It was sprayed with 2% ferric
chloride in ethanol solution. A positive result
was seen during the experiment in this test
which is the formation of bright blue spot as
displayed in Figure 2.
Test for Flavanoids
The spotted plate was placed in a beaker
containing ethyl acetate, glacial acetic acid
and water in a 90:10:20 ratio respectively.
The plate was removed and air dried after
the solvent has reached the top of the plate.
It was sprayed with 5% ferric chloride
solution. The formation of gray spot is taken
to be a positive result as shown in Figure 3.

Test for Saponins

A solution of chloroform and methanol in a
30:5 ratio was placed in a beaker. The
spotted plate was then placed in that
beaker. It was removed after the solvent has
reached the tip of the plate. It was air-dried.
A solution of vanillin sulfuric acid was placed
in the sprayer and the plate was sprayed
with it. The formation of violet spots as
displayed in Figure 4 was taken to be a
positive result in this test.
Test for Tannins
A solution of chloroform, methanol and
water in a 66:35:10 ration was prepared and
placed in a beaker. The spotted plate was set
in that beaker and it was removed and airdried after the solvent has spread up to the
top of the plate. A solution of 0.5% (w/w)
vanillin in 4% (w/w) HCl was used as the
spraying solution. After the spotted plate
was sprayed, it showed a negative result as
shown in Figure 5. Supposedly, the positive
result for this is the formation of blue spots.

Figure 1. Test for Alkaloids

Figure 2. Test for Phenols

Figure 4. Test for Saponins

Figure 3. Test for Flavonoids

Figure 5. Test for Tannins

Results and Discussion

Phytochemicals are secondary metabolites
in one or more parts of the medicinal plants.
These have the ability to produce a definite
physiological action on the human body. In
the experiment, the extract from Euphorbia
hirta plant were analyzed qualitatively. Table
1 presents the results of phytochemical
analysis.
Table 1. Phytochemical Screening
Test

Result

Alkaloids

Negative

Phenols

Positive

Flavonoids

Positive

Saponins

Positive

Tannins

Negative

Phenols, flavonoids and saponins were

found in the extract of Euphorbia hirta. This
is in accordance with the results of the study
of Patil and Magdum (2011). They have
found out that the ethanolic extract of
Euphorbia hirta showed a positive result for
phenols, steroids, cardiac glycosides,
saponins, carbohydrates and flavonoids.
The presence of these secondary
metabolites in Euphorbia hirta, produce
some biological activity in man and animals
and it is responsible for their use as herbs.
These compounds also serve to protect the
plant against infection by microorganisms,
predation by insects and herbivores (Ketkar
and Ketkar, 1995).
Conclusions and Recommendations
The goal of this experiment was to screen
the ethanolic extract of Euphorbia hirta for

the presence of various secondary

metabolites. It was found out that the
ethanolic extract of the plant contains
phenols, saponins and flavonoids.
Since the results are in accordance with
previous study conducted, the experiment
itself is successful in screening the ethanolic
extract of the plant. Further studies should
be conducted especially in isolating the
compounds found in the plant.
References
Arts, I.C. and Hollman P.C. (2005).
Polyphenols and disease risk in
epidemiologic studies. Am J Clin Nutr,
81(1 Suppl): p. 317S-325.
Ketkar, A.Y. and Ketkar, C.M. Various uses of
nem products: Medicinal uses inducing
pharmacology in Asia in H. Schmuterer,
Ed. 1995, p. 518-525.
Park, J. B. (2006). Phytochemical Database.
Retrieved from the world wide web:
http://www.ars.usda.gov/News/docs.h
tm?docid=8875
Pastil, S.B. and Magdum C.S. (2011).
Phytochemical
Investigation
and
antitumour activity of Euphorbia hirta
Linn. European Journal of Experimental
Biology, 1(1): 51-56.