JOMO KENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
DEPARTMENT OF BIOCHEMISTRY
HBB 2150: INTRODUCTION TO BIOCHEMISTRY
PRACTICAL 1 - QUALITATIVE TESTS FOR CARBOHYDRATES
Various carbohydrates can be identified by carrying out tests which are specific for the various
carbohydrate structures. In the experiments which follow you are going to identify these various
carbohydrate structures in following solutions;
1. Solution A 2. Solution B 3. Solution C 4. Solution D
Procedure
Test 1: Molish’s Test
Mix 2ml of each solution and add 5 drops of molish;s reagent in a test tube. Add gently through
the side, 2ml of concentrated sulphuric acid (H₂SO₄). The acid forms the lower layer. Observe
the purple ring at the interface of layers. The furfural formed by the dehydration of the sugar by
the acid condenses with the naphthol in the reagent to give the coloured ring. This test is general
for all carbohydrates. Which of the solutions is not a carbohydrate?
Test 2: Benedicts Test
Put 3ml Benedict’s solution, in separate test tube, add 8 drops of each of the solutions. Mix and
keep in boiling water for 5 minutes. A precipitate shows a reduction by the sugar. What are your
observations?-
Test 3: Seliwanoff’s Test
Put 3ml of the reagent in separate test tubes, add 10 drops of each of the solutions. Boil and cool.
Fructose gives a red colour.
, JOMOKENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
DEPARTMENT OF BIOCHEMISTRY
HBB 2150: INTRODUCTION TO BIOCHEMISTRY
PRACTICAL 2: QUANTITATIVE DETERMINATION OF GLUCOSE IN URINE US-
ING BENEDICT’S METHOD
The principle carbohydrate in the organism are hexoses composed of six carbon atoms such as
glucose and fructose and the pentose’s or five carbon atom sugars. The hexoses supply a large
part of the energy required by the cell and can be metabolized to pentoses which are important
part of nucleic acid molecules. Carbohydrates can be classified as polyhydroxy compounds hav-
ing either aldehyde group (aldoses) and ketone group (ketoses). These will reduce certain metal
ions with the sugar undergoing oxidation in the reaction forming a number of low molecular
weight compounds. This reaction is the basis of Benedict’s test for reducing sugars and quantita-
tive determination of glucose in body fluids.
Benedict’s reagent for the estimation of reducing sugars contains potassium thiocynate as well as
copper sulphate. In the former white precipitate of cuprous oxide, the small amount of potassium
ferrocynide also aids in keeping cuprous oxide in solution. As the precipitate white is formed, the
loss of all blue tints in the solution indicates complete reduction of the copper.
Procedure
1. Place 3ml of the quantitative Benedict’s reagent with a pipette into test tube.
2. Place the test tube on heating block and bring to boiling.
3. On boiling, add drop wise to the solution (with 2.0 ml graduated measuring cylinder),
your unknown two solutions (Sample A and B). Start with your measuring cylinder filled
to the 10ml mark. Add drop wise till a brown precipitate forms, then continue to boil for
2 minutes and then add very slowly some further drops until the benedicts colour has en-
tirely disappears.
4. Make a note of the amount of the sample used.
DEPARTMENT OF BIOCHEMISTRY
HBB 2150: INTRODUCTION TO BIOCHEMISTRY
PRACTICAL 1 - QUALITATIVE TESTS FOR CARBOHYDRATES
Various carbohydrates can be identified by carrying out tests which are specific for the various
carbohydrate structures. In the experiments which follow you are going to identify these various
carbohydrate structures in following solutions;
1. Solution A 2. Solution B 3. Solution C 4. Solution D
Procedure
Test 1: Molish’s Test
Mix 2ml of each solution and add 5 drops of molish;s reagent in a test tube. Add gently through
the side, 2ml of concentrated sulphuric acid (H₂SO₄). The acid forms the lower layer. Observe
the purple ring at the interface of layers. The furfural formed by the dehydration of the sugar by
the acid condenses with the naphthol in the reagent to give the coloured ring. This test is general
for all carbohydrates. Which of the solutions is not a carbohydrate?
Test 2: Benedicts Test
Put 3ml Benedict’s solution, in separate test tube, add 8 drops of each of the solutions. Mix and
keep in boiling water for 5 minutes. A precipitate shows a reduction by the sugar. What are your
observations?-
Test 3: Seliwanoff’s Test
Put 3ml of the reagent in separate test tubes, add 10 drops of each of the solutions. Boil and cool.
Fructose gives a red colour.
, JOMOKENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
DEPARTMENT OF BIOCHEMISTRY
HBB 2150: INTRODUCTION TO BIOCHEMISTRY
PRACTICAL 2: QUANTITATIVE DETERMINATION OF GLUCOSE IN URINE US-
ING BENEDICT’S METHOD
The principle carbohydrate in the organism are hexoses composed of six carbon atoms such as
glucose and fructose and the pentose’s or five carbon atom sugars. The hexoses supply a large
part of the energy required by the cell and can be metabolized to pentoses which are important
part of nucleic acid molecules. Carbohydrates can be classified as polyhydroxy compounds hav-
ing either aldehyde group (aldoses) and ketone group (ketoses). These will reduce certain metal
ions with the sugar undergoing oxidation in the reaction forming a number of low molecular
weight compounds. This reaction is the basis of Benedict’s test for reducing sugars and quantita-
tive determination of glucose in body fluids.
Benedict’s reagent for the estimation of reducing sugars contains potassium thiocynate as well as
copper sulphate. In the former white precipitate of cuprous oxide, the small amount of potassium
ferrocynide also aids in keeping cuprous oxide in solution. As the precipitate white is formed, the
loss of all blue tints in the solution indicates complete reduction of the copper.
Procedure
1. Place 3ml of the quantitative Benedict’s reagent with a pipette into test tube.
2. Place the test tube on heating block and bring to boiling.
3. On boiling, add drop wise to the solution (with 2.0 ml graduated measuring cylinder),
your unknown two solutions (Sample A and B). Start with your measuring cylinder filled
to the 10ml mark. Add drop wise till a brown precipitate forms, then continue to boil for
2 minutes and then add very slowly some further drops until the benedicts colour has en-
tirely disappears.
4. Make a note of the amount of the sample used.
