IDENTIFICATION OF PHYTOCONSTITUENTS

Dr.U.Srinivasa, M.Pharm,Ph.D

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.

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.

(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.

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.

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.

(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.

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.

(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.

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 TESTTo 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.

(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.

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.

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.

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.

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 oilTESTS FOR FIXED OIL AND FATS 1. Press the extract in between the two filter papers , a

permanent stain indicates the presence of fixed oil2. 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

GUMS AND MUCILAGE'S1. Powder is treated with few drops of Ruthenium

red solution , the particles acquires pink colour2. 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

ESTIMATION OF PHYTOCONSTITUENTS

ALKALOIDS

GLYCOSIDES

TANNINS

VOLATILE OILS

RESINS

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

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.