1.2.

Classification Tests

1.1.1. Reducing and non-reducing sugars

Benedict’s test:-
Objective: -

To determine whether the sugar is a reducing or a non-reducing

Material and method:-

1- In a clean, dry test-tube put 2 ml of the available sugar

solution (or: few crystals = 2 ml of distilled water)

2- Add 2 ml of Benedict’s reagent and shake gently.

3- Heat in a boiling water-bath for 2 minutes.

4- Observe and record the changes.

Observations: -

Red colour (or: red precipitate).

Results:

The presence of the red colour (Range: green-yellow-pink-red)

indicate that the sugar is reducing (positive).

Explanation:-

Benedict’s reagent contains copper sulphate (Cu++) which

reduced by the reducing sugar (Reducing sugars are

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 carbohydrates that have free aldehyde or ketone groups) to
insoluble red copper oxide (Cu+) (which seen as precipitated).

Benedict’s = CuSO4 + Sodium citrate + Sodium carbonate.

CuSO4 + 2 NaOH Cu (OH) 2 + Na2SO4

Blue

CHO

(CH─OH)n + Cu (OH) 2 Cu2O ↓+ H2O +

CH2OH Red

Aldose

COOH

(CH─OH)n

CH2OH

Aldonic acid

2
 Before boiling

After boiling for 2 minutes

1.2.2. Aldoses and Ketoses Sugars

Seliwanoff's test:

Objective: To determine whether the sugar is an aldose or a ketose.

Material and Methods:-

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 1- Put 0.5 ml of in a clean test-tube, add 3 ml of Seliwanoff's
reagent and shake well.

2- Place in a boiling water-bath for exactly 2 minute.

3- Observe and record your observation.

Observation:-

Red colour (or: pink) with ketoses.

Results:-

Red colour indicates the presence of the Ketonic group.

Explanation:-

This test depends on the same idea of Anthrone’s test which is

the formation of function of furfural and hydroxy- methyl
furfural by losing 3 molecules of water because of the presence
of the concentrated acid (HCl). The formation of furfural and
hydroxyl methyl furfural is more readily in ketoses than aldoses
which then condense with resorcinol to form red complex.

Hexose Hydroxymethyl furfural

Resorcinol Red
complex

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

1.2.3. Test for Pentoses

Bial's Test

Objective: To distinguishes a pentose (5 carbons) from a hexose (6

carbons) monosaccharide.

Method:

1. Add 2 ml of sugar solution to be tested to 5 ml of Bial's reagent.

2. Gently heat the tube to boiling.

3. Allow the tube to cool.

4. Observe and record your observation.

Observation:

Formation of a green color solution or precipitate.

Results:

Formation of a green colored solution or precipitate indicates a positive reaction.

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Explanation:-

Bial’s reagent contains orcinol in concentrated HCl with a small amount of

Fe3Cl catalyst. Pentoses react with Bial’s reagent to form furfural, which
condenses with orcinol to produce a blue-green product.
OH
O

OH

orcinol furfural

Two negative tests (left, middle) and a positive test (right)

1.2.4. Test for Starch

Iodine test:-

Objective: - To determine the presence of starch

Material and method:-

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 1- Add few drops of iodine solution to 2 ml of the available sugar in a
clean test-tube.

2- Observe and record the results.

3- Into another test-tube put 2 ml of starch.

4- Add 1 ml of conc. HCL and heat for 10 minutes then adds few
drops of iodine solution.

Observations: -

1. Dark violet colour (or blue –black).

2. After heating no dark violet (blue-black colour).

Results:-

A polyiodide complex is formed.

Explanation:-

1. Starch composed of amylopectin and amylose chains and the

later is helix in its structure. Helical structure of amylose chain
make iodine accumulates inside it and form a complex of
amylose-iodine complex.

2. As starch is broken down or hydrolyzed into smaller

carbohydrate units, the blue-black colour is not produced.
Therefore, this test can determine completion of hydrolysis
when a colour change does not occur.

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Left to right: iodine, starch solution, starch solution + iodine.

Results: Yellow-orange = negative.

Violet colour or blue-black = positive.