highlights upon the important chemical tests routinely conducted for the identification of important phytoconstituents

1. Mohini Upadhye
Department of Pharmacognosy
PES Modern college of Pharmcy (For
Ladies), Moshi, Pune
Identification of phytoconstituents

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