ANSWERED LABORATORY EXPERIMENT # 4

A. Molisch’s Test

Reagents used:
1. H2SO4
2. Molisch reagent (Component: a. Ethanol (C2H5OH) b.5% α-naphthol (C10H8OH)

Purpose of the test:

 to detect carbohydrates (monosaccharides, disaccharides and polysaccharides) and glycoprotein
in an analyte.
 can be used to differentiate proteins and amino acids from carbohydrates
Principle:
o concentrated sulfuric acid (H2SO4) is added to the analyte which hydrolyzes all the glycosidic linkage in the
sugar molecules (disaccharide, and polysaccharide) to yield monosaccharide.
o When monosaccharides are treated with concentrated H2SO4, hydroxyl (OH) groups of sugar are removed
in the form of water, and
o Furfural is formed from pentose sugar.
o Hydroxymethylfurfural is formed from hexose sugar (e.g., glucose).
o These reactive products condense with α-naphthol to give a purple/violet colored complex

Solution Presence of Violet Ring

Glucose +
Lactose +
Sucrose +
Starch +
Furfural -
B. Action of Alkali
Reagents used:
1. 10% NaOH
2. 10% Ba(OH)2

Purpose of the test:

o Detect reducing sugars
o Knok how strong and weak base react with carbohydrates/ reducing sugars.

Principle:
o Strong alkali decomposes sugars like strong acids. Weak alkali acts upon sugars.
o The disaccharides are easily affected by acid, and they are the least readily decomposed by alkali.
Positive Reaction:
o Formation of brown to black char

1. 10% NaOH solution

Solution Color and Odor produced

Glucose BROWN-BLACK CHAR / BURNT-SUGAR SMELL
Fructose BROWN-BLACK CHAR / BURNT-SUGAR SMELL
Galactose BROWN-BLACK CHAR / BURNT-SUGAR SMELL

2. 1% Ba(OH)2 solution

Solution Color and Odor produced

Glucose LIGHT BROWN CHAR / LESS BURNT-SUGAR
SMELL
Fructose LIGHT BROWN CHAR / LESS BURNT-SUGAR
SMELL
Galactose LIGHT BROWN CHAR / LESS BURNT-SUGAR
SMELL

C. Reduction Tests
Reducing sugars are carbohydrates having free aldehyde or ketone functional group in its molecular structure. All
monosaccharides are reducing sugars because they either have an aldehyde group (if they are aldoses) or can
tautomerize in solution to form an aldehyde group (if they are ketoses). This includes common monosaccharides
like galactose, glucose, glyceraldehyde, fructose, ribose, and xylose.
A. Fehling’s Test

Fehling’s solution composition:

1. CuSO4 (Copper sulphate)
2. KNaC4H4O6·4H2O (Potassium sodium tartrate also known as Rochelle salt)
Purpose of the Test:
 for the determination of reducing and non-reducing sugars that are present in carbohydrates

Principle:
o When aldehydes are added to Fehling’s solution, they are easily oxidized by the bistartratocuprate (II)
complex. During this process, copper (II) ions get reduced to copper (I) ions leaving a red precipitate of
copper (I) oxide (Cu2O)
o The presence of red precipitate indicates a positive result

Solution Presence of reddish brown precipitate

Glucose +
Fructose +
Lactose +
Maltose +
Sucrose -
Starch -

Sucrose is a non-reducing sugar because the two monosaccharide units are held together by a glycosidic
linkage between C1 of α-glucose and C2 of β-fructose. Since the reducing groups of glucose and fructose are
involved in glycosidic bond formation, sucrose is a non-reducing sugar.
B. Benedict’s Test
Benedict’s solution composition: 1. CuSO4. 5H2O (copper sulfate pentahydrate)
2. Na3C6H5O7 (sodium citrate)
3 Na2CO3 (sodium carbonate)
4. distilled water
Purpose of the Test:
o to detect reducing sugars

Principle:
o The presence of the alkaline sodium carbonate converts the sugar into a strong reducing agent called
enediols. During the reduction reaction, the mixture will change its color from blue to brick-red
precipitate due to the formation of cuprous oxide (Cu2O).
o Copper in its cupric (Cu2+) or copper (I) form is reduced to cuprous (Cu+) or copper (II).
o The red-colored cuprous oxide is insoluble in water and hence, separated.
o If the concentration of the sugar is high, then the color becomes more reddish, and the volume of the
precipitate increases

Solution Presence of brick-red precipitate

Glucose +
Lactose +
Sucrose -
Starch -

C. Barfoed’s Test

Barfoed’s reagent: cupric acetate in 1% acetic acid

Test solutions/Samples:
1 % Glucose, 1 % Fructose, 1 % Lactose, 1 % Maltose, 1 % Sucrose, 1 % Unknown sugar

Purpose of the Test:

 used for detecting the presence of reducing sugars/monosaccharides.

Shows positive test for: Reducing monosaccharides

Principle of Barfoed’s test:
Barfoed’s test is used for distinguishing monosaccharides from reducing disaccharides. Monosaccharides
usually react in about 1-2 minute while the reducing disaccharides take much longer time between 7-12
minutes to react with the reagent. Brick red color is obtained in this test which is due to formation of
cuprous oxide.
 Reactions:
Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid and a
reddish precipitate of copper (I) oxide within three minutes. Reducing disaccharides undergo the same
reaction, but do so at a slower rate.

A positive test is indicated by:

The formation of a reddish precipitate within three minutes.

D.

a negative test (left) and a positive test (right)

 Positive Barfoed’s test: development of brick red color ppt within 3-5 minutes
 Negative Barfoed’s test: absence of red color
** reducing disaccharides also give positive barfoed test on prolong heating

Solution Presence of reddish precipitate

Glucose + (first 3 minutes)
Fructose + (first 3 minutes)
Lactose -
Maltose -
Sucrose -
Unknown sugar -

E. Nylander’s Test
Purpose of the Test:
- used for detecting the presence of reducing sugars/monosaccharides.
Nylander's reagent:
1. Bi(NO3)3 - (bismuth nitrate)
2. KNaC4H4O6·4H2O - (potassium sodium tartrate)
3. KOH - (potassium hydroxide)

Test solutions/Samples: 1 % Glucose, 1 % Fructose, 1 % Maltose, 1 % Lactose

Principle of Nylander’s test:

A test for glucose using a solution containing bismuth subnitrate which forms a black precipitate in a
positive reaction. When Nylander's reagent is added to a solution with reducing sugars, a black
precipitate of metallic bismuth is formed.

In the case of alkaline solution (KOH), bismuth sub nitrate, the bismuth hydroxide formed is reduced to
 black metallic bismuth as shown in the following equation:

Bi(OH)2NO3 + KOH --> Bi(OH)3 + KNO3

2Bi(OH)3 (with reducing sugar and heat) --> 2Bi + 3O + 3H2O

A positive test is indicated by:

The formation of a black precipitate. The conclusion of the test is that when sugar is present, there is a
brown to black colouration of the fluid where the metallic bismuth settles down. If the dark coloration
occurs as the fluid is cooling down, it does not prove the presence of sugar.

Solution Presence of Black precipitate

Glucose +
Fructose +
Lactose +
Maltose +

F. Picric Acid Test

Reagents:
1. C6H3N3O7 Saturated picric acid/2,4,6-trinitrophenol (TNP)
2. Na2CO3 Sodium Carbonate (10% solution)

Purpose of the Test: one of the tests for the detection of reducing sugars.

Principle:
o The picric acid test for carbohydrates is a very sensitive chemical test for the presence of
reducing sugars. The reducing sugars react with Picric Acid (toxic yellow crystalline solid) also
chemically known as 2,4,6-trinitrophenol (TNP) to form a red coloured Picramic Acid.
o All monosaccharides and disaccharides containing the potentially free aldehyde or ketone
group possess reducing properties.
o Reducing sugars contains a free aldehyde or ketone group possess reducing property. They
reduce some organic acids when in alkaline solution.
o The Sodium Carbonate (Na2CO3) is added to make the solution alkaline or basic.
o Thus, reducing sugars reduce picric acid(yellow solution) to picramic acid (mahogany red
solution).

Reaction:
 Interpretation:
 Appearance of red colour indicates the presence of reducing sugar in the solution.

Solution Presence of Black precipitate

Glucose +
Fructose +
Lactose +
Maltose +

G. Tollens Test
Reagents:
Tollens Reagent refers to the chemical reagent which is used in the detection of an aldehyde functional
group, an aromatic aldehyde functional group, or an alpha hydroxy ketone functional group in a given
test substance.

The Tollens Reagent is named after Bernhard Tollens, A German chemist who discovered this reagent
and its uses. Tollens reagent is a solution of silver nitrate (AgNO3) and Ammonia (NH3).

Purpose of the Test:

Tollens’ test, also known as silver-mirror test, is a qualitative laboratory test used to distinguish between
an aldehyde and a ketone. It exploits the fact that aldehydes are readily oxidized (see oxidation),
whereas ketones are not.
Principle:
The Tollen’s test is used in organic chemistry to test for the presence of aldehydes. In this reaction,
an aldehyde is oxidized to a carboxylic acid while the Ag1+ is reduced to silver metal, which deposits as
a thin film on the inner surface of the glass. The generic reaction is as follows and is specific for
aldehydes:
Reactions:

The silver ions present in the Tollens reagent are reduced into metallic silver.

Generally, the Tollens Test is carried out in clean test tubes made of glass. This is because the
reduction of the silver ions into metallic silver form a silver mirror on the test tube. This silver mirror is
illustrated in the example below.

Figure 11: Tollens' test for aldehyde: left side positive (silver mirror), right side negative

Tollens test is commonly referred to as the Silver Mirror test due to the formation of this layer of metallic silver
on the test tube.

Ketones are not oxidized by Tollens’ reagent, so the treatment of a ketone with Tollens’ reagent in a glass
test tube does not result in a silver mirror (Figure 11; right).