Experiment 6: Qualitative tests for sugars and amino acids

Aim:
To identify the presence of sugar and amino acid given samples

Molisch’s Test:
Developed by Hans Molisch to confirm for the presence of carbohydrates Upon addition of one
drop of sulphuric acid or hydrochloric acid to a given carbohydrate solution, it gets
dehydrated to produce an aldehyde called 5-hydroxy methyl furfural, which condenses with two
molecules of phenol or (α-naphthol) (add one drop) resulting in a red (with phenol) - or purple-
colored (α-naphthol) compound at the junction of two liquids which confirms the presence of
carbohydrates

Iodine Test for starch and Glycogen:

To detect the presence of polysaccharides like starch and glycogen upon addition of Iodine gives
blue, black color, glycogen gives red, orange hazy color and dextrin gives red color. Starch
consists of two fractions amylase (α (1→4) bound glucose molecules 300-3000) and amylopectin
(α (1→4) bound glucose molecules with α (1→6) branch points at every 24-30 glucose units)

Example:

Tests for Monosaccharide’s, Disaccharides and reducing sugars:

1. Barfoeds Test: Invented by Christen Thomsen Barfoed.

It is used to detect the presence of monosaccharide (reducing) sugars in solution.
Barfoed's reagent is prepared with a 0.33 molar solution of neutral copper acetate in
1% acetic acid solution.
 Acetic acid and copper (II) acetate is added into the test solution and boiled.
A brick red copper (I) oxide precipitate is formed, which indicates the presence of
reducing sugar. Reducing Disaccharides like maltose, Lactose, cellobiose, etc. may react,
but they are much slower.
The reaction will be negative in the presence of disaccharides like sucrose, Trehalose,etc
because they are not reducing agents.
This test is highly specific for reducing monosaccharide and less specific to reducing
disaccharides. Due to the weakly acidic nature of Barfoed's reagent, it is reduced quickly
by monosaccharide.

RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+

2. Benedicts Test: Invented by Stanley Rossiter Benedict for detecting reducing sugars.
Benedict's test is that when reducing sugars are heated in the presence of an alkali;
they get converted to powerful reducing compounds known as enediols. Enediols reduce
the cupric ions (Cu2+) present in Benedict's reagent to cuprous ions (Cu+) which
get precipitated as insoluble red copper (I) oxide (Cu2O).

The difference between Barfoeds and Benedicts is the later can be used for disaccharide
reducing sugars like lactose and maltose. It is a semi-quantitative test with different colors
with the amount of reducing sugar present in the test sample.

Green precipitate denotes 0.5 % (w/v) reducing sugar; yellow precipitate denotes 1%
(w/v) of reducing sugar; orange denotes 1.5 % (w/v) reducing sugar and red denotes > 2 %
(w/v) of reducing sugar.

Alkali
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+

Example:

3. Osazone Test:
Invented or discovered by Emil Fisher when he was working with the
stereochemistry of sugars.

When the sugars are reacted with an excess of phenyl hydrazine they give rise to
respective sugar osazone crystals, the reaction is used to identify monosaccharide’s, which
give needle or broom-shaped crystals whereas disaccharides give the puffy ball or
sunflower like radiating crystals. Two phenyl hydrazine molecules react with the ‘α-’
Carbon, because of its more reactivity and followed by subsequent carbon as shown in the
figure.

Glucose Phenyl hydrazine Glucoosozone

Procedure

Note: “+” sign refers to addition of the next chemical to the previous step.

A. Molish test:

1. To 2 ml of the test solution + 100 µl of Molish reagent and mix it well.

2. Incline the tube and add 1 ml of concentrated sulfuric acid (carefully) along the sides
of the tube.
3. Observe the delicate red and violet colour ring which appears at the junction of the
two liquids.
4. Note your results in the observation table.

B. Barfoed’s test

1. Take 500 µl of test solution + add 500 µl of barfoed’s reagent.

2. Keep it in the boiling water bath for a minute.
3. Allow to cool in the stand for few minutes.
4. Observe the formation of brick-red precipate (indicate mono saccharides).
5. Note your results in the observation table.

C. Iodine test:

1. Add 100 µl of iodine solution to 1 ml of test solution.

 2. Observe the appearance of deep blue coloured ring which indicates the presence of
starch in the solution.
3. Note your results in the observation table.

D. Benedict test:

1. To 2 ml of the Benedict’s reagent, add 200 µl of test solution.

2. Keep it in the boiling water bath for 5 minutes.
3. Allow to cool in the stand for few minutes.
4. Observe theformation of red, yellow or green colour precipate, which indicates
presence of reducing sugars.
5. Note your results in the observation table.

E. Osazone test

1. Take 0.5 gram of phenyl hydrazine hydrochloride, to this add 0.1g of sodium acetate.
2. Add 10 drops of glacial acetic acid.
3. Add 5 ml of test solution.
4. Keep it in boiling waterbath for 30 minutes.
5. Allow it to cool for a while.
6. Examine the crystals under microscope:
For glucose and fructose- needle shaped yellow asazone can be observed
For lactose – mushroom/cotton ball shape can be observed
For maltose – flower-shaped crystal can be observed

Observation table:

Reagent Positive Negative Conclusion Unknown 1 Unknown 2

control control
Molish Test Glucose BSA(Protein) Carbohydrate is
present
Barfoed Test Glucose Starch Monosaccharide
present
Iodine Test Starch Glucose Polysaccharide
present
Benedict Test Glucose Sucrose Reducing sugar
present
Osazone Test Glucose Not Needle shaped Not
applicable yellow crystals applicable
Lactose Mushroom/cotton
ball shape
Maltose Flower-shaped
crystals