Qualitative chemical examination of extracts

Qualitative chemical examination of extracts

The extraction can be done by taking powdered drugs in

The general method followed for screening of the constituents present in the extract is as follows:-------------------------------------------------------------------------------------------------

Detection of carbohydrates:

Extracts were dissolved individually in 5 ml of distilled water and filtered. The filtrates were used to test the presence of carbohydrates.

    a)   Molisch’s test: (named after Austrian botanist Hans Molisch) Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube, and 2ml concentrated sulphuric acid was added carefully along the sides of the test tubes. The formation of a violet ring at the junction indicates the presence of carbohydrates.

    b) Benedict’s test (also called Benedict's solution or Benedict's test) is a chemical reagent named after an American chemist, Stanley Rossiter Benedict) (One liter of Benedict's reagent can be prepared from 100 g of anhydrous sodium carbonate, 173 g of sodium citrate, and 17.3 g of copper(II) sulfate pentahydrate. It is often used in place of Fehling's solution.)

Filtrates were treated with Benedict’s reagent and heated in a water bath. The formation of an orange-red precipitate indicated the presence of reducing sugars.

C)    Fehling’s test: The test was developed by German chemist Hermann von Fehling in 1849.^[2] Fehling's solution is always made just prior to the test. It is composed of equal parts of the following solutions:

• 69.28 grams copper(II) sulfate pentahydrate dissolved in 1 liter of distilled water (Fehling's solution A)
• 346 grams Rochelle salt (potassium sodium tartrate tetrahydrate) and 120 grams sodium hydroxide in 1 liter of distilled water (Fehling's Solution B)

Filtrates were hydrolyzed with dilute hydrochloric acids, neutralized with alkali, and heated with Fehlings A and B solutions. The formation of a red precipitate indicates the presence of reducing sugars.

d) Osazone formation: A sugar, on heating with Phenyl hydrazine hydrochloride, sodium acetate, and acetic acid, forms yellow crystals of osazone.

e)  Selivanoff’s test: (Resorcinol test for ketoses )  A crystal of resorcinol is added to the solution and heated with an equal volume of concentrated hydrochloric acid  Pink color is produced in the case of ketoses (eg. Fructose, honey, or hydrolyzed insulin).

f) Test for pentose: A solution of the material is heated with an equal volume of hydrochloric acid containing a little phloroglucinol. A red color is formed in the case of pentose.

g) Furfural test: The sample is heated in a test tube with a drop of syrupy phosphoric acid to convert it into furfural. A disk of filter paper moistened with a drop of 10% solution of aniline in 10% acetic acid is placed over the mouth of the test tube. The bottom of the test tube is heated for 30-60 seconds. A pink or red stain appears on the reagent paper.

h)  Barfoed's Test: The Barfoed's Test is a chemical test carbohydrates that detects the presence of monosaccharides. Placing one ml of a sample solution in a test tube, 3 ml of Barfoed's reagent, a solution of acetic acid, and cupric acetate solution are added. Heat the solution in a boiling water bath for three minutes, and if a formation of reddish precipitate appears, it tests positive for monosaccharides.

Detection of alkaloids: Extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrates were tested carefully with alkaloid reagents.

a) Mayer’s test: Filtrates were treated with Mayers reagent (potassium mercuric iodide), formation of a yellow cream precipitate indicates the presence of alkaloids.

b) Wagner’s test: Filtrates were treated with Wagner’s reagent (iodine in potassium iodide) and observed. The formation of a brown/reddish brown precipitate indicates the presence of alkaloids.

c) Dragendroff’s test: Filtrates were treated with Dragendroff’s reagent (solution of potassium bismuth iodide). The formation of a red precipitate indicates the presence of alkaloids.

d) Hager’s test: Filtrates were treated with Hager’s reagent (saturated picric acid solution) Formation of a yellow-colored precipitate indicates the presence of alkaloids.

e)  Tannic acid test: A freshly prepared solution of tannic acid (55w/w) gives a precipitate with most of the alkaloids, which is soluble in dilute acid or ammonia solution.

f) Ammonia Reineckats test: A saturated aqueous solution of ammonia reineckate, slightly acidified with hydrochloric acid, gives a pink flocculent precipitate with most of the alkaloids.

Detection of phytosterols:

a) Salkowski’s test: The extracts were treated with chloroform and filtered separately. The filtrates were treated with a few drops of concentrated sulphuric acid, shaken, and allowed to stand. If the lower layer turns red, sterols are present. If the lower layer turns golden yellow triterpenes are present.

b) Liebermann Burchard test: The extracts were treated with chloroform solution and a few drops of conc. 1 ml of acetic anhydride solution followed by sulphuric acid. A blue-green colour shows the presence of phytosterols.

Detection of glycosides:

Extracts were hydrolyzed with dilute hydrochloric acid, and the hydrolysate was subjected to glycoside tests.

Test for cardiac glycosides:

   

 a. Legals test: To the hydrolysate, 1 ml of pyridine and a few drops of sodium nitroprusside solution are added, and then it is made alkaline with sodium hydroxide solution. Colour change shows the presence of glycosides.

b. Liebermann’s test: 3 ml. of extract with 3 ml. acetic anhydride was heated, cooled then a few drops of conc. sulphuric acid. The blue color appears in the presence of bufadenolide.

c. Test for deoxysugars (Keller killiani test): 2 ml. of extract, glacial acetic acid, one drop of 5% FeCl3, and conc. H2SO4 was added. A reddish-brown color appears at the junction of two liquid layers, and the upper layer appears bluish-green.

d. Xanthydrol test: The crude drug is heated with a 0.1 to 5% solution of Xanthydrol in glacial acetic acid containing 1% hydrochloric acid. A red color is produced due to the presence of 2-deoxysugar.

Test for anthraquinone glycosides:

a. Borntrager’s test: Hydrolysate is treated with chloroform, and the chloroform layer is separated. To this, an equal quantity of dilute ammonia solution is added. Color change in the ammoniacal layer shows the presence of O glycosides.

b. Modified Borntrager’s Test: The extracts were treated with ferric chloride solution and heated in a boiling water bath for about 5 min. The mixture was cooled and shaken with an equal volume of benzene. The benzene layer was separated and treated with half its volume of ammonia solution. The formation of pink or cherry red color in the ammonical layer indicates the presence of a C glycoside.

Detection of saponin:

a) Froth’s test: The extracts were diluted with distilled water to 20 ml, shaken in a graduated cylinder for 15 min. The formation of a 1 cm layer of foam indicates the presence of saponins.

b) Haemolytic test: Add drug extract or dry powder to one drop of blood placed on a glass slide. A hemolytic zone appears.

Test for Cyanogenic glycosides:

Ferriferrocyanide test: Macerate 1 g of the powdered drug with 5 ml of alcoholic KOH for 5 min. transfer it to an aqueous solution containing FeSO₄ and FeCl₃, and maintain at 60⁰-70⁰C for 10 minutes. Now transfer of the contents confirms the presence of HCN.

Detection of phenolic and tannins:

a. Ferric chloride test: The extract was treated with a few drops of neutral ferric chloride solution. The formation of a bluish-black color indicates the presence of a phenolic nucleus.

b.Gelatin test: To the extract, 1% gelatin solution containing sodium chloride was added. The formation of a white precipitate indicates the presence of tannins.

c. Goldbeater’s test: A small piece of goldbeater skin (membrane prepared from the intestine of an ox) is soaked in 20% Hydrochloric acid, rinsed with distilled water, and placed in a solution of tannins for 5 minutes. The skin pieces are washed with distilled water and kept in a solution of ferrous sulphate. A brown or black color is produced due to the presence of tannins.

d. Phenazone test: A mixture of aqueous extract of a drug and sodium acid phosphate is heated, cooled, and filtered. A solution of phenazone is added to the filtrate. A bulky-colored precipitate is formed.

e. Match stick test (Catechin test): a match stick is dipped in aqueous plant extract, dried near a burner, and moistened with concentrated hydrochloric acid. On warming near the flame, the matchstick wood turns pink or red due to the formation of phloroglucinol.

Test for flavonoids:

a. Lead acetate test: The extracts were treated with a few drops of lead acetate solution; the formation of a yellow precipitate indicates the presence of flavonoids.

b. Alkaline reagent test: The extracts were treated with a few drops of sodium hydroxide separately. The formation of an intense yellow color, which becomes colorless with the addition of a few drops of dilute acid, indicates the presence of flavonoids.

c. Shinoda test: The extracts were treated with a few fragments of magnesium metal separately, followed by a dropwise addition of concentrated hydrochloric acid. The formation of a magenta color indicates the presence of flavonoids.

Detection of proteins and amino acids:

a. Millon's test: (The reagent is made by dissolving metallic mercury in nitric acid and diluting it with water. The test was developed by the French chemist Auguste Millon (1812–67).

The extracts were treated with 2ml of Millon's reagent. The formation of a white precipitate, which turns to red upon heating, indicates the presence of proteins.

b. Ninhydrin test: (Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is produced. Ninhydrin is most commonly used to detect fingerprints, as the terminal amines or lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin.) 

To the extracts, 0.25% ninhydrin reagent was added and boiled for a few minutes. The formation of blue color indicates the presence of amino acids.

c. biuret test: (The biuret reagent is made of potassium hydroxide (KOH) and hydrated copper (II) sulfate, together with potassium sodium tartrate. The reagent turns from blue to violet in the presence of proteins, and blue to pink when combined with short-chain polypeptides.) 

An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most often), followed by a few drops of aqueous copper(II) sulfate. If the solution turns purple, protein is present. 5–160 mg/mL can be determined.

d.