Glycosides on account of their varied nature are first analyzed for the nature of the sugar and then tested according to the characteristic of the aglycone. Many glycosides are coloured, fluorescent, or chromogenic and are therefore tested accordingly. Different chemical tests specific for the aglycone are used to detect them after hydrolysis.

I. Some of the general tests for glycosides are:

1. A solution of glycoside gives a deep red colour with concentrated sulphuric acid.
2. To a pinch of the substance dissolved in glacial acetic acid, add a few drops of ferric chloride solution followed by concentrated sulphuric acid. Appearance of a red ring at the junction of two layers indicates the presence of glycosides.

II. Tests specific for sugars in glycosides

1. Molisch’ test: To a pinch of the drug dissolved in water add 1 ml hydrochloric acid and heat to boiling. Cool and treat with α-naphthol and a few drops of concentrated sulphuric acid. Formation of a purple colour indicates the presence of a glucoside, a glucose containing glycoside.
2. Treat about 0.2 g of the crude drug with 5 ml dilute sulphuric acid and warm on a water bath for 2 minutes. To the supernatant add equal volumes of Fehling’s solution A and Fehling’s solution B and heat for 2 minutes. A brick-red precipitate indicates the presence of glycosides.
3. Keller-Kiliani test: Dissolve a pinch of the glycoside in 3 ml glacial acetic acid containing 2 drops of 5% ferric chloride. Carefully transfer this to the surface of 2 ml concentrated sulphuric acid. A reddish-brown layer forms at the junction of the two layers and the upper layer slowly becomes bluish-green and darkens on standing. This test is specific for glycosides containing desoxy sugars
4. Paper chromatography for the identification of sugars
   A solution of the hydrolyzed mixture of the glycoside is spotted on a paper along with marker sugars. It is developed in the upper layer of butanol-acetic acid-water (4:1:5) system. After a run of about 24 hours, air dry the paper, and spray with aniline hydrogen phthalate solution. Sugars are seen as visible brown-coloured spots. Comparable R_f of the sugar with that of the authentic samples will indicate the sugar present in the glycoside.

III. Tests specific for aglycones

1. Cardiac glycosides answer keller-kiliani test. In addition they answer tests for the presence of the unsaturated lactone ring and tests for steroids. (See under “Identification of digoxin” (Chap 8, p. 284).) Strophanthus glycosides reduce tollen’s reagent and they also give a precipitate with tannic acid.
2. Tests for anthraquinones
   1. Born Trager’s test: Boil 0.1 g of the drug with 2 ml dilute sulphuric acid and centrifuge. Pipette off the supernatant, cool and shake with an equal volume of carbon tetrachloride and separate it. Treat 1 ml of it with half its volume of dilute ammonia. A rose-red colour is formed in the ammoniacal layer indicating the presence of anthraquinones. To another 1 ml of the organic layer add methanolic magnesium acetate solution; an orange colour is formed.
   2. Modified Born Trager’s test: Boil 0.1 g of the drug with 1 ml dilute hydrochloric acid and 2 ml of 5% aqueous ferric chloride solution. Filter while hot, cool, and shake the filtrate with carbon tetrachloride. Separate the organic layer and shake it with 2 ml dilute ammonia. A rose-red colour is formed in the ammoniacal layer. C-glycosides such as carcarosides may be identified by this modified test as they are resistant to normal acid hydrolysis and require oxidative hydrolysis with ferric chloride for the cleavage of the sugar–nonsugar bond.
3. Tests for triterpenes and flavonoids discussed under saponin and flavonoid glycosides.
4. Tests for cyanogenetic glycosides: Moisten a pinch of the drug with water and add a small amount of dilute sulphuric acid. Heat the flask gently and as heating progresses suspend a strip of filter paper previously moistened and dried with sodium picrate solution into the test tube clamped between the test tube cork closure. Hydrocyanic gas is released as a result of hydrolysis and this turns the picrate paper to a red colour.