EXPERIMENT 1/ QUALITATIVE ANALYSIS of CARBOHYDRATES:

The essential preliminary of biochemistry is to develop an appreciation of the chemical

structure and properties of those substances of which living systems are composed.
Carbohydrates make up the bulk of organic substances on earth. They are an important
source of carbon compounds and chemical energy either for immediate use in such
compounds as glucose or fructose or for storage in the form of polysaccharides such as
starch or glycogen. Many carbohydrates, such as cellulose and chitin, serve a structural
function. Structural polysaccharides are frequently found in combination with proteins
(glycoproteins or mucoproteins) or lipids (lipopolysaccharides). For classification purposes,
carbohydrates may be divided into three subgroups; monosaccharides, oligosaccharides,
and polysaccharides.

Carbohydrates are vital for our life; this experiment aims to introduce you with the
identification of unknown carbohydrates. To gain maximum benefit, observations should
be related, as far as possible, to the structure of the substances examined.

Work systematically; initial observations should be made on solubility and first tests should
be those for the classification of compounds, e.g. Molisch’s test for carbohydrates. You
will find it useful to device an analytical scheme for yourself.

Some important points:

1. Most of the tests and reactions described are not quantitative and volumes are
approximate, despite these facts some tests do not work if quantities greatly in
excess of those stated are used. Do not place your pipettes in reagent bottles as this
leads to contamination.

2. In most tests it is important to apply a control test using water instead of the
solution under examination. If you are in doubt about the result of a test, perform
the reaction with a suitable known compound.
TESTS ON CARBOHYDRATES:

Molisch’s Test:

All carbohydrates, and some compounds containing carbohydrates in a combined form, are
degraded by concentrated H2SO4, either to furfural or a derivative.

⇒ Apply this test to two carbohydrate solutions of your own choice, preferably to one
monosaccharide and one polysaccharide.

Procedure:
- To 2ml of a known solution add 1 drop of Molisch’s reagent (10% α-naphthol in ethanol).
- Down the side of a sloping tube slowly pour 1-2ml of conc. H2SO4, so that it forms a
layer at the bottom of the tube.
- Observe the color at the interface between two layers.
- Perform a control test with water instead of a sugar solution.

• A brown color due to charring must be ignored and the test should be repeated with
a more dilute sugar solution.
• A green color should also be ignored.
• This test is positive for all carbohydrates, free or combined. Many proteins also give
this reaction, since they may contain carbohydrate groups.
™ What is the reaction that leads to the formation of a furfural derivative?
™ Please include structures and reactions in your reports!

Solubility:

⇒ Apply this test to all the carbohydrates provided.

Check solubility both in water and alcohol.

1. Carbohydrates as Reducing Agents:

All mono and disaccharides with a free aldehyde or keto group act as reducing agents in
alkaline solutions. The reducing properties of sugars are dependent upon the presence of
actual or potential aldehyde or ketone groups.
The enolization of sugars under alkaline conditions is an important consideration in
reduction tests. The ability of a sugar to reduce alkaline test reagents depends on the
availability of an aldehyde or keto group for reduction reactions. A number of sugars,
especially disaccharides or polysaccharides have glycosidic linkages which involve bonding
between each group, and hence there is no reducing group on the sugar; such as the case
for sucrose, trehalose, inulin, glycogen, starch, and dextrin. In the case of reducing sugars,
the presence of alkali causes extensive enolization especially at high pH and temperature.
This leads to a higher susceptibility to oxidation reactions than at neutral or acidic pH.
These sugars, therefore, become potential agents capable of reducing Cu+2 to Cu+, Ag+ to
Ag and so forth. Reducing sugars can react with many different oxidizing agents. Fehling’s
test, Benedict’s test and Barfoed’s test have been used to distinguish between
monosaccharides and disaccharides

a-Fehling’s Test:

⇒ Perform this test with fructose, glucose, maltose and sucrose

Procedure:
- To 1ml of Fehling’s solution A (aqueous solution of CuSO4) add 1ml of Fehling’s solution
B (solution of sodium potassium tartrate)
- Add 2ml of the sugar solution, mix well and boil.

• Try to see the red precipitate of cuprous oxide that forms.

™ What is the function of tartrate?
™ Some disaccharides such as maltose are reducing agents, whereas others, such as
sucrose are not. Explain why?

b-Barfoed’s Test:

Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so that it
can only be reduced by monosaccharides but not by less powerful reducing sugars.

⇒ Perform this test with glucose, maltose and sucrose.

Procedure:
- To 1-2ml of Barfoed’s reagent, add an equal volume of sugar solution.
- Boil for 5min. in a water bath and allow to stand.

• You will observe a cuprous oxide precipitate if reduction has taken place.

c-Seliwanoff’s Test:

Ketoses (e.g. fructose) are more readily decomposed by acid than the more stable aldoses.
In this reaction, HCl is used instead of H2SO4, and resorcinol is used as the chromogen.

⇒ Perform this test with glucose, fructose, maltose and sucrose.

Procedure:
- Heat 1ml of sugar solution with 3ml Seliwanoff’s reagent (0.5gr resorcinol per liter
10%HCl) in boiling water. Note the proportions.
- In less than 30seconds a red color must appear for ketoses.

• Upon prolonged heating glucose will also give an appreciable colour.

d-Bial’sTest:

This test is utilized for the identification of pentoses.

⇒ Perform this test to ribose and glucose.

Procedure:
- To 5ml of Bial’s Reagent (resorcinol in conc. HCl) add 2-3 drops of ribose solution and
boil
- Note the green blue color formed.

2. Action of Alkali on Sugars:

Procedure:
- Heat 1ml glucose solution with 1ml 40% NaOH for 1min.
- Cool and apply test for reducing sugars

™ Explain the reaction of glucose and NaOH.

3. The Inversion of Sucrose:
Procedure:
- Add 5ml of sucrose solution to two tubes.
- Add 5 drops of conc. HCl to one tube
- Heat both tubes in a boiling water bath for 10min.
- Cool and neutralise with dil. NaOH (use litmus)
- Test both solutions for the presence of reducing sugar with Fehling’s test

™ Explain the result.

™ What is an invert sugar?
™ What is “inversion”?
™ What would you expect from a similar reaction with starch?
™ How would you identify the product of hydrolysis?

4. Iodine Test:

⇒ Apply this test to all the polysaccharides provided.

Procedure:
- To 2-3ml of the polysaccharide solution add 1-2 drops of iodine solution.