Qualitative Phytochemical Investigation (1)

All the extracts were subjected to preliminary qualitative phytochemical analysis to test for the presence of various phytoconstituents like alkaloids, phenolics, saponins, carbohydrates etc. For this purpose fresh phytochemical reagents were prepared. Detection of Alkaloids 1.1 Mayers Test To a few ml of extract, add a drop or two of Mayers reagent (1.358 g of Mercuric chloride in 60 ml of water mixed with 5 gm potassium iodide in 10 ml of water and made up to 100 ml with water) along the side of the test tube. A white or creamy precipitate indicates the test as positive. 1.2 Wagners Test To a few ml of extract, add few drops of Wagners reagent (1.27 g of Iodine & 2 gm Potassium iodide dissolved in 5 ml water and made up to 100 ml with water) along the sides of the test tube. A reddish-brown precipitate confirms the test as positive. 1.3 Hagers Test To a few ml of extract, add 1 or 2 ml of Hagers reagent (saturated solution of Picric acid in water). A prominent yellow precipitate indicates the test as positive. 1.4 Dragendroffs Test To a few ml of extract, add 1 or 2 ml of Dragendroffs reagent. A prominent yellow precipitate indicates the test as positive.

Dragendroffs reagent Stock solution : Boil 5.2 gm Bismuth carbonate and 4 gm sodium iodide for a few minutes with 50 ml glacial acetic acid. After 12 hours, filter off the precipitated sodium acetate crystals and mix 40 ml of the clear, red-brown filterate with 160 ml ethyl acetate and 1 ml water. Stored in amber colored glass bottle. Working solution: 10 ml of stock solution mixed with 20 ml of acetic acid and made up to 100 ml with water. 2 Detection of Carbohydrates 2.1 Molischs Test To 2 ml of extract, add two drops of Molish reagent (alcoholic solution of Napthol) and shake well. Then add 1 ml concentrated sulfuric acid along the sides of the test tube and allow standing. A violet ring indicates the presence of carbohydrates. 2.2 Fehlings Test 1 ml extract boiled with 1 ml each of Fehlings solutions A and B. A red precipitate indicates the presence of sugar. 2.3 Barfoeds Test To 1 ml of extract, 1 ml of Barfoeds Reagent (30.5 gm Copper acetate monohydrate dissolved in 1.8 ml glacial acetic acid made up to 100 ml with water). Red precipitate indicates the presence of sugar. 2.4 Benedicts Test

To 1 ml of extract, add 1 ml Benedicts reagent (Boil 17.3 gm of Sodium citrate and 10 gms Sodium carbonate in 80 ml of distill water to get a clear solution. Add 1.73 gms copper sulphate in 10 ml water to the above solution.) A characteristic colored precipitate indicates the presence of sugar. 3 Detection of Saponins 3.1 Foam Test 50 mg extract dissolved in 20 ml of distilled water, shaken for 15 min in a graduated cylinder. A 2 cm layer of foam indicates the presence of saponins. 4. Detection of Proteins & Amino Acids 4.1 Millons Test To 2 ml of extract, add few drops of Millons reagent (1 gm Mercury dissolved in 9 ml fuming Nitric acid. After completion of reaction 9 ml of water was added). A white precipitate indicated the presence of proteins. 4.2 Biurets Test 2 ml of extract treated with one drop of 2 % Copper sulphate solution. Then add 1 ml of 95% Ethanol, followed by excess of Potassium hydroxide. Pink color in ethanolic layer indicated the presence of protein. 4.3 Ninhydrin Test To 2 ml of extract, add 2 3 drops of Ninhydrin solution (10 mg of ninhydrin in 20 ml Ethanol). A characteristic purple color indicates the presence of proteins. 5 Detection of Steroids 5.1 Salkowaskis test

When a few drops of conc. H2SO4 are added to the test solution, shaken and allowed to stand, lower layer turned red indicated the presence of sterols. 5.2 Liebermann Burchard test The test solution treated with few drops of acetic anhydride. When concentrated Sulphuric acid is added from the sides of the test tube, it showed a brown ring at the junction of the two layers and upper layer turned green. 6 Detection of Phenolic Compounds & Tannins 6.1 Ferric chloride Test Dissolve 50 mg extract in 5 ml of water and add few drops of neutral 5% Ferric chloride solution. A dark green color indicates the presence of phenolic compounds. 6.2 Lead Acetate Test To 2 ml of extract add 1 ml of 10% Lead acetate solution. A bulky white precipitate indicates the presence of phenolic compounds. 6.3 Vanillin HCl Test To 2 3 ml of extract add few drops of Vanillin HCl reagent (Dissolve 1 gm Vanillin in 10 ml Ethanol and add 10 ml concentrated HCl). A red color showed presence of phenolic compounds.