Table 1 Methods followed in qualitative biochemical tests

Glycoside

0.5 mL of sample were evaporated and dissolved in 1 mL glacial acetic acid. 1 drop of 10% FeCl₃ solution followed by 1 mL of Conc. H₂SO₄ was added by the side of test tube. Appearance of brown colour rings at the interface would indicate the presence of glycosides.

Glycerol

Dunstan’s test was used for identification of glycerol. Phenolphthalein drops in borax solution give pink colour. If addition of a sample to this solution leads to the disappearance of pink colour in cold which however reappears on heating and again disappears on cooling would indicate the presence of glycerol.

Phenol

In 2 mL of sample, 200 µL of Folin–Ciocalteu reagent and 2 mL 20% (w/v) Na₂CO₃ were added. Appearance of blue colour would indicate the presence of phenol.

Flavonoid

Alkaline reagent test was done here. Two drops of NaOH solution were added to 2 mL of sample. Initially, a deep yellow colour appeared but it gradually became colourless by adding few drops of dilute HCl, indicating that flavonoids were present.

Coumarin

Few drops of NaOH solution was added to 1 mL of sample. Yellow coloration would indicate the presence of coumarin

Tannin

To 0.5 mL sample and few drops of HNO₃ was added. The reddish to yellow colour of the solution would indicate the presence of tannins.

Fatty acid

Few drops of phenolphthalein solution was added into a test tube followed by two drops of diluted NaOH solution. A pink colour will appear. Few drops of sample was then added and shaken. A disappearance of pink colour would indicate the presence of fatty acids in the sample.

Protein

Biuret test was followed here. Two drops of 3% CuSO₄ and few drops of 10% NaOH were added to 1 mL of sample, violet or red colour formation would indicating the presence of proteins.

Terpenoid

Horizon test was followed here. Two millilitres of trichloroacetic acid was added to 1 mL of sample. The presence of terpenoids was confirmed by the formation of a red precipitate.

Steroid

For test of 0.5 mL samples were evaporated and dissolved in 2 mL chloroform. 2 mL of concentrated H₂SO₄ was introduced carefully by the sidewall of the test tube. Formation of red colour ring would confirm the presence of steroid.

Alkaloid

Dragendorff ’s test was done. By adding 1 mL of Dragendorff”s reagent to 2 mL of sample, an orange red precipitation would indicate the presence of alkaloids.