AIM: To Perform identification test for monosaccharides
References: 1. S.P. Singh, Practical Manual of Biochemistry, CBS Publishers and Distributors, New
Delhi, Fifth Edition, 19-37.
2. R. Chawla, Practical Clinical Biochemistry: Methods and Interpretations, Jaypee brother's medical
publishers (P) LTD. New Delhi, Third Edition, 32-38.
Requirements:
Glass-wares: Test tubes, Test tubes holder, Beaker, Water bath and Glass rod.
Chemicals: Sample solution, Molisch reagent, Conc. Sulphuric acid, lodine solution, Benedict's
reagent, Fehling reagent, Barfoed's reagent, Seliwanoff's reagent, Sodium carbonate solution (10%),
Phenyl hydrazine hydrochloride, Sodium acetate, Glacial acetic acid.
Introduction:
Carbohydrates are polyhydroxy aldehydes and ketones or substances that hydrolyse to yield
polyhydroxy aldehydes and ketones. They serve as the primary source of energy. Carbohydrates are
mainly divided into monosaccharides, disaccharides and polysaccharides. The commonly occurring
monosaccharide includes glucose, fructose, galactose, ribose etc. The two monosaccharides
combined together to form disaccharides which include sucrose, lactose and maltose. Starch and
cellulose fall into the category of polysaccharides which consists of many monosaccharide residues.
Experimental procedure:
1. Molisch test
Procedure: To take 2 ml of test solution in a test tube and add two drops of Molisch's reagent (alpha-
napthol) and mix well Incline the test tube and add about 1 mL of concentrated sulphuric acid along
the sides of the test tube. Observe the colour at the junction of the two Liquide
Observation: A purple-violet ring appears at the junction of the two liquids.
Inference: Carbohydrate present.
2. Charring test:
Procedure: Carbohydrates on heating in a test tube or in presence of Conc. H₂SO
Observation: Produces charring with smell like burning sugar.
Inference: Carbohydrate present.
3. lodine Test
Procedure: take 2 ml. of the test solution in a test tube and add a few drops of iodine solution.
Observation: Absence of blue colour.
Inference: This indicates the absence of starch (polysaccharides) in the solution.
4. Benedict test
Procedure: To take 5 drops of the test solution in a test tube and add 2 mL of Benedict's reagent. Boil
for five minutes in a water bath. Cool the solution
Observation: Formation of red, yellow or green colour on basis of concentration of glucose in test
sample.
Inference: This indicates the presence of reducing sugars in the test solution may be present.
5. Fehling test
Procedure: To take 1 ml of Fehling's solution 'A' add 1 mL of Fehling's solution 'B' and a few drops of
the test solution in a test tube. Boil for a few minutes.
Observation: Formation of brownish-red yellow or precipitate
Inference: This indicates the presence of reducing sugars in the test solution (Glucose may be
present).
6. Barfoed test
Procedure: take 2 mL of the test solution in a test tube and add about 2 mL of Barfoed's reagent. Boil
it for one minute and allow to stand for a few minutes
Observation: Formation of brick-red precipitate.
Inference: Only monosaccharides answer this test (Glucose may be present).
7. Seliwanoff test
Procedure: take 2 ml of test solution in a test tube and add 2 ml of reagent. Heat the mixture to just
boiling.
Observation: Appearance of deep red colour.
Inference: Ketoses sugar may be present (Glucose may be absent).
Remark: Seliwanoff's test distinguishes between aldose (glucose) and ketose sugars. A ketohexose
like fructose, sucrose will form a deep red Seliwanoff's reagent while an aldohexose will show a light
pink takes a longer time to develop the colour. No colour change in case of test sample as glucose.
8. Ozasone test
Procedure: To take 0.5 g of phenyl hydrazine hydrochloride, 0.1 g of sodium acetate and 10 drops of
glacial acetic acid in a test tube To this mixture add 5 mi of test solution and heat on a boiling water
bath for about half an Allow the test tube to cool slowly and examine the crystals under the
microscope.
Observation: Formation of crystals
Inference: Glucose produces needle-shaped yellow ozasone crystals
9. Bial’s test
Procedure: Bial's test is a qualitative test used to differentiate between pentoses and hexoses in
carbohydrates. The test uses Bial's reagent, which contains orcinol, ferric chloride, and hydrochloric
acid. 2ml of test solution is mixed with 2ml of reagent.
Observation: muddy brown colour was observed due to presence of glucose.
Inference: A blue or green color indicates the presence of pentoses.
A muddy-brown, yellow, or gray color indicates the presence of hexoses.
Conclusion for tests: all above tests are for monosaccharides.
