Preliminary phytochemical screening of aqueous extract.

 Experiment

Object

To perform Preliminary phytochemical screening of  aqueous extract.

Requirements:

Glassware: Test tube, glass rod, dropper,test tube stand, porcelain dish etc

Chemicals: Crude drug power/extract, chloroform, alcohol,Mayer's reagent, Wagner's reagent,Dragondroff's reagent, Hager's reagent, Benedict's reagent, Fehling's reagent, ninhydrin reagent,

Theory:

 The various chemical tests were performed on this extract and aqueous extract for the 

identification of flavonoids, phenolic compounds, alkaloids, glycosides, carbohydrates, 

carotenoids, proteins, tannin, aminoacids, sterols as per Harborne 1998.

Extraction of crude drug

1.Take 50 gm of powdered crude drug and macerate it with 500 ml of water for 24 

hrs.

2.Then occasionally shake with 6hr time period and allow it to stand for 18 hr

3. After filtration evaporate the filtrate to dryness in a tare flat bottom shallow dish

Preparation of test solution

4. Take 500 mg of extract and dissolve it in 100 ml of water. stir the solution till the 

extract is completely soluble in water.

5.The sample solution is then subjected to various qualitative test to reveal the 

presence or absence of common phytopharmaceuticals.

TEST FOR ALKALOIDS

About 2 gm of the powdered material was mixed with 1gm of calcium hydroxide and 5 

mL of water into a smooth paste and set aside for 5 minutes. It was then evaporated to dryness in 

a porcelain dish on a water bath. To this 200 mL of chloroform was added, mixed well and

refluxed for half an hour on a water bath. Then it was filtered and the chloroform was

evaporated. To this 5 mL of dilute hydrochloric acid was added followed by 2 mL of each of the 

following reagents.

MAYER’S TEST:

A small quantity of the extract was treated with Mayer’s reagent. Cream colour 

precipitate indicates the presence of alkaloids.

DRAGENDORFF’S TEST:

A small quantity of the extract was treated with Dragendorff’s reagent. Orange brown 

precipitate indicates the presence of alkaloids.

WAGNER’S TEST:

A small quantity of extract was treated with Wagner’s reagent. Reddish brown precipitate 

indicates the presence of alkaloids.

HAGER’S TEST:

A small quantity of extract was treated with Hager’s reagent. Yellow precipitate indicates

the presence of alkaloids

TEST FOR PURINE GROUP (MUREXIDE TEST):

The residue obtained after the evaporation of chloroform was treated with 1mL of 

hydrochloric acid in a porcelain dish and 0.1 gm of Potassium chlorate was added and 

evaporated to dryness on water bath. Then the residue was exposed to the vapour of dilute 

ammonia solution. No purple Colour was obtained indicating the absence of purine group of alkaloids.

TEST FOR INDOLE

To the test solution, add acetic acid and trace amount of anhydrous FeCl3, under

– lay /H2SO4 intense blue at interface.

TEST FOR QUINOLINE (Thalleioquin Test)

To the extract, add 1 drop of dilute sulphuric acid and 1ml of water. Add bromine water drop 

wise till the solution acquires permanent yellow colour and add 1mL of dilute ammonia solution, 

emerald green colour is produced. The powdered drug when heated with glacial acetic acid in 

dry test tube, evolves red fumes, which condense in the top portion of the tube. The bark, when 

moistened with sulphuric acid and observed under ultraviolet light shows a blue fluorescence 

due to the methoxy group of quinine and quinidine.

TEST FOR CARBOHYDRATES 

MOLISCH’S TEST

The extract of the powdered drug was treated with 2-3 drops of 1% alcoholic α naphthol 

and 2mL of concentrated sulphuric acid was added along the sides of the test tube. A purple 

colour indicating the presence of carbohydrates.

FEHLING’S TEST

The extract of the powdered leaf was treated with Fehling’s solution I and II and heated 

on a boiling water bath for half an hour. Red precipitate was obtained indicating the presence of free reducing sugars.

TEST FOR PROTEIN AND AMINO ACIDS

MILLON’S TEST

Small quantity of acidulous – alcoholic extract of the powdered drug was heated with 

Millon’s reagent. White precipitate turned red on heating indicate the presence of proteins.

BIURET TEST

To one portion of acidulous – alcoholic extract of the powdered drug one ml of 10% 

sodium hydroxide solution and one drop of dilute copper sulphate solution were added. Violet colour was obtained indicating the presence of proteins.

NINHYDRIN TEST

To the test solution add Ninhydrin solution, boil, violet colour indicates presence of 

amino acid.

TEST FOR SULPHUR CONTAINING AMINO ACID:

5 ml test solution is mixed with 2 ml 40 % sodium hydroxide and 2 drops of 10% lead 

acetate solution. Then boil the solution turned black or brownish due to PLS formation.

TEST FOR TERPENOIDS

Little of the powdered drug was extracted with chloroform and filtered. The filtrate was 

warmed gently with tin and thionyl chloride. Pink colour solution appeared which indicated the presence of terpenoids.

Observation:

Make observation table on blank page.

Result:

Preliminary phytochemical screening of the aqueous extract of (Sample Name)shows 

the presence of___________________