1. IDENTIFICATION OF
PHYTOCONSTITUENTS
Dr.U.Srinivasa, M.Pharm,Ph.D

2. TESTS FOR STEROIDS
SALKAOWSKI TEST
To the chloroform solution in a test tube concentrated
sulphuric acid was added from the sides of the test tube.
A reddish brown color was observed.
LIEBERMAN BURCHARDT TEST
To the chloroform solution in a test tube a few drops of
acetic anhydride was added and mixed well.1ml of
concentrated sulphuric acid was added from the sides of
the test tube and allowed to stand. A reddish ring was
formed at the junction of two layers.

3. TESTS FOR TRITERPENES
(a) SALKAOWSKI TEST
A few drops of concentrated sulphuric acid were added to
the chloroform solution, shaken and allowed to stand. Lower
layer turned yellow.
(b) LIEBERMAN BURCHARDT TEST
To the chloroform solution a few drops of acetic anhydride
and 1ml of concentrated sulphuric acid was added. A deep
red color was produced.

4. (C)TRICHLORO ACID AND STANNIC CHLORIDE
TEST
To the chloroform solution a few drops of thionyl chloride
and a pinch of stannic chloride were added. A range of
colors green, blue, purple and finally turning to red were
obtained.
(D) KAHLENBERG TEST
To 0.2 ml of the chloroform solution a few drops of
antimony pentachloride and chloroform was added. A
deep purple color was observed.

5. TESTS FOR SAPONINS
FOAM TEST : Small amount of extract was shaken in a test
tube with a little quantity of water, the foam produced
persisted for 10 minutes. This confirms the presence of
saponins.
TESTS FOR LACTONES
(a) LEGAL’S TEST : To the extract mixture of sodium nitropruside
and pyridine was added. The mixture was treated with
sodium hydroxide. It gave a deep red color.
(b) BAL JETS TEST : To the various extracts treated with
sodium picrate solution. Yellow to orange color was
produced shows the presence of lactone ring.

6. TESTS FOR ALKALOIDS
(A) MAYER’S TEST
The various extracts were dissolved in chloroform.The
chloroform was evaporated and the residue was acidified
and added few drops of Mayer’s reagent (Potassium Mercuric
Iodide). A creamy white precipitate was observed.
(B) WAGNER’S TEST
The various extracts were dissolved in chloroform. The
chloroform layers were evaporated, to the residue were
acidified and added few drops of Wagner’s reagent (Iodine in
Potassium Iodide). Orange precipitate was observed.


7. (C) DRAGENDROFF’S TEST
The various extracts were dissolved in chloroform. The
chloroform was evaporated and the residue was acidified and
added few drops of Dragendroff’s reagent (Potassium
Bismuth Iodide). Orange red precipitate was observed.
(D) HAGER’S TEST
The various extracts were dissolved in the chloroform. The
chloroform was evaporated and the residue was acidified and
added few drops of Hager’s reagent (Saturated Picric Acid
solution). Yellow crystalline precipitate was observed.

8. TEST FOR CARBOHYDRATES
(A) MOLISCH’S TEST
The extract with Molisch’s reagent mix and added
concentrated sulphuric acid along the sides to form layers. A
reddish violet ring at the interference shows the presence of
carbohydrates.
(B) FEHLING’S TEST
The extract was heated with Fehling’s A and B solution it
gave an orange red precipitate shows the presence of
reducing sugar.

9. (C) BENEDICT’S TEST
With Benedict’s reagent the carbohydrates on boiling and
cooling a green reddish brown precipitate was formed which
shows the presence of reducing sugar
(D) BARFOEDS TEST
To the extract Barfoeds reagent was added and it was
boiled on a water bath, reddish precipitate was observed
within 90 minutes show the presence of monosaccharide.

10. TEST FOR FLAVANOIDS:
(A) FERRIC CHLORIDE TEST
To a small quantity of the alcoholic solution of the extract a
few drops of neutral ferric chloride solution was added. A
green color was produced due to the phenolic nucleus.
(B) SHINODA TEST
To the alcoholic solution of the extract a few fragments of
magnesium ribbon were added. To this concentrated
hydrochloric acid was added drop wise. Magneta color was
produced after few minutes which are the characteristic
reaction of flavanoid.

11. (C) ZINC-HYDROCHLORIDE ACID REDUCTION TEST
To the alcoholic solution of the extract a pinch of zinc dust
was added and few drops of concentrated hydrochloric acid
were added. Magneta color was produced after a few
minutes.
(D) LEAD ACETATE TEST
To the alcoholic solution of the extract few drops of lead
acetate solution (10%) was added. Yellow precipitate was
observed.

12. TEST FOR TANNINS:
(A) FERRIC CHLORIDE TEST
2 ml of extract was taken in a test tube and ferric chloride
solution was added drop by drop. A blue black precipitate
was observed.
(B) GELATIN TEST
To the extract few drops of 1% solution of gelatin containing
10% sodium chloride was added. A white precipitate was
observed. These tests confirmed the presence of tannins.

13. TEST FOR PROTEINS:
(A) BIURET TEST
Reagent 40% sodium hydroxide and dilute copper sulphate
solution. Protein shows blue, pink or violet color where as
amino acids fails to show the color. The extract does not
give this reaction. Shows the absence of protein and amino
acids.
(B) NINHYDRIN TEST
With Ninhydrin amino acids show blue color. But proteins
may give the positive tests very rarely. The extract does not
give the Ninhydrin reaction.

14. TEST FOR GLYCOSIDES:
(A) BAL JETS TEST
To the various extracts with sodium picrate solution. Yellow
to orange color was observed which shows the presence of
glycoside with lactones ring.
(B) KELLER-KILLANI TEST
To the various extracts 1ml of glacial acetic acid and few
drops of ferric chloride solution was added and then slowly
concentrated sulphuric acid was added through the sides of
the test tube. A reddish brown ring at the junction of liquids
was observed which shows the presence of de-oxysugar.

15. TESTS FOR VOLATILE OILS
1. Volatile oil is soluble in alcohol (90%)
2. Put one drop of volatile oil on the filter paper, no
permanent stain indicates the presence of volatile oil
TESTS FOR FIXED OIL AND FATS
1. Press the extract in between the two filter papers , a
permanent stain indicates the presence of fixed oil
2. Extract is treated with few drops of 0.5N potassium
hydroxide and few drops of phenolphthalein and heat ,
formation of soap indicates the presence of fixed oil and fats

16. GUMS AND MUCILAGE'S
1. Powder is treated with few drops of Ruthenium red solution ,
the particles acquires pink colour
2. Powder when treated with water or aqueous KOH , the
particles swells
3. Aqueous extract with few drops of dilute Hcl and equal
volumes of Fehling's solution A and B and heat, red colour is
produced indicates the presence of gums

17. ESTIMATION OF PHYTOCONSTITUENTS
 ALKALOIDS
 GLYCOSIDES
 TANNINS
 VOLATILE OILS
 RESINS

18. GLYCOSIDES
CARDIAC GLYCOSIDES :
1. By colorimetric method
2. By biological assay

19. THE DIFFERENCE BETWEEN A SUCCESSFUL PERSON
AND OTHERS IS NOT A LACK OF STRENGTH, NOT A
LACK OF KNOWLEDGE, BUT RATHER A LACK IN WILL.