Rating: ________

Name: JULKANAIN, AMINAH D.

Section: BSMT 2A

Activity No. 2

CARBOHYDRATES
I.OBJECTIVES:

1. To detect the presence of Monosaccharide, Disaccharides and

Polysaccharides using different tests.

II.PROCEDURES:
a. Using the internet visit an educational website and search for the procedure,
chemical composition and positive results of the following test;
b. Write the procedure and attach an image for the positive results.

A. MONOSACCHARIDES AND DISACCHARIDES

1. Moore’s Test

Procedures:
I. Mix 1ml of 5% glucose solution with 1 ml concentrated NaOH.
II. Boil and note the change of colour and the odour produced
Principles:

This test is based on the liberation of aldehydes which subsequently polymerize

to form a resinous substance, caramel.

Chemical Composition:
Concentrated Alkali (NaOH)
Positive Results:

In Moore’s test the colour of the

solution changes from
colourless to caramel.
 2. Molisch’s Test

Procedures:

I. Place 1ml of 5% of glucose solution in a test tube.

II. Add 1 drop of Molisch’s reagent and mix thoroughly
III. Incline the tube and allow 1ml concentrated sulphuric acid to flow on the
side of the test tube (do not shake)
IV. Place the tube on the rack and note the colour produced at the junction of
two liquids.

Principles:

Molisch Test is a general test of carbohydrates and any compound containing

a carbohydrates residue in the molecule. The test is sensitive and a dilution as
low as .001% still gives a positive result.

Chemical Composition:
A-napthol reaction and sulphuric acid
Positive Results:

The colour that was

produced at the junction of
the two liquids is a brown-
yellow colour and formed
furfural derivatives

B. REDUCTION TEST

1. Fehling’s Test

Procedures:

I. Place 5 ml each of Fehling's A and B in a test tube.

II. Dilute it with 4 cc of water.
III. Boil 1 ml of this mixture in a water bath. (If change of color is observed,
it indicates contamination. Discard and get a freshly prepared solution).
 If color is unchanged after boiling, add 5% glucose solution drop by
drop hearing the mixture after each dilution.
IV. Note the changes produced.
Principles:

The Fehling’s test is the solution changes its color but at the end of the
boiling process, it turns back to its original color with red precipitate.

Chemical Composition:
Fehling’s A (CuS04) and water; Fehling's B (KOH); Rochelle Salt (sodium
potassium tartrate) and water.

Positive Results:

From a blue
solution it changed
to green the yellow
and lastly to
brownish-red
precipitate.

2. Benedict’s Test

Procedures:

I. Mix 1ml of Benedict’s reagent with 2 drops of glucose solution.

II. Boil for 2 minutes and allow cooling.
III. Observe for any changes of colour
Principles:

The Benedict’s test is a test to determine whether or not the carbohydrate

contains a free aldehyde or ketone group

Chemical Composition:
Sodium Citrate, anhydrous sodium carbonate and water.

Positive Results:
 From its original
blue solution it
changed into
green, tallow,
orange, red and
brick red
3. Nylander’s Test

Procedures:

I. Mix 1ml of 5% glucose with .1ml of Nylander’s reagent

II. Heat for 5 minutes in a water bath and note the results.
III. Write the reaction involved in the test. Nylander’s reagent: Bismuth sub
nitrate, Rochelle salt and 19% KOH
Principles:

Nylander’s test is a chemical test used for detecting the presence of reducing
sugars. When Nylander’s reagent, consists of bismuth nitrate, potassium sodium
tartrate and potassium hydroxide, is added to a solution with reducing sugars, a
black precipitate of metallic bismuth is formed.

Chemical Composition:
Bismuth sub nitrate; Rochelle salt and 19% KOH
Positive Results:

The Colour of Nylander’s

reagent with 1ml of 5%
glucose is originally a
clear solution and
changes to yellow,
orange and dark brown

4. Barfoed’s Test

Procedures:

I. Mix 1ml of Barfoed’s reagent with .1% of glucose solution.

II. Heat for 30 seconds and allow to stand for 15 minutes
Principles:

Barfoed’s test can distinguish monosaccharides from di- and polysaccharides

because with the conditions of lower PH
 Chemical Composition:
Cupric acetate and Acetic acid solution

Positive Results:

The phosphomolybdenum
blue formed causes a deep
blue coloration in the
solution in the
monosaccharide solution.

5. Picric Acid Test

Procedures:

I. To 1ml of 5% glucose solution add .5ml of saturated picric acid solution and .
2ml of 10% Na2CO2
Principles:

The Picric acid test is a test for carbohydrates which is very sensitive for
chemical test in the presence of reducing sugars. The reducing sugars react with
picric acid turns into toxic yellow crystalline solid, also known as 2, 4,6-
trinitrophenol (TNP) to form a red coloured picramic acid.

Chemical Composition:
Glucose Solution, saturated picric acid and Na2CO3
Positive Results:
 The color was light
yellow to dark yellow
into red color

6. Seliwanoff’s Test

Procedures:

I. Place 1ml of seliwanoff’s reagent in each of 6 test tubes.

II. To the first tube, add 3 drops of 2% freshly prepared solution in fructose’s, to
the second add glucose, to the third add galactose, to the fourth add
sucrose, to the fifth add maltose, and to the sixth add lactose.
III. Boil in a water bath
IV. Note the colour produced and record the time required for the
development of pink in each tubes.

Principles:

This test is useful for detecting the presence of Kete-hexose. It is due to the
conversion of fructose (by HCL) into levulinic acid and hydroxylmethylfrlfural
which condenses with resorcinol to form a red color. Other sugars, especially
sucrose will also give a positive result test, but usually require a longer time

Chemical Composition:
HCL and resorcinol non-oxidizing acid.
Positive Results

The cherry-red complex is

formed in the reaction
indicating the presence of
the ketose sugar

7. Bial’s Orcinol Test

Procedures:

I. Place a 2ml of sample solution in a test tube.

II. Add 2ml of Bial’s reagent which is a solution of orcinol, HCL and ferric
chloride.
III. Then Heat the solution gently in a Bunsen burner/ water bath
IV. Observe the solution, if the colour is not obvious, add more water
Principles:

The Bial’s test is used to distinguish pentose sugar

Chemical Composition:
HCL orcinol and ferric chloride
Positive Results:

The condensation reaction of pentose

with orcinol will Form a green yellow
colour change in the solution. Hexoses
may also form a color change, but
these changes from red to brown.
Green color change indicates the
presence of pentose sugar

8. Mucic Acid Test

Procedures:
I. Place 1ml of 6% carbohydrate solution in each of the test tubes
II. Add 1ml of concentrated HNO3
III. Then heat in a boiling bath for 1 to 2 hours
IV. Remove the tubes and observe the result
Principles:

This test is one in which concentrated HNO3 is heated along an aldose sugar to
give a dicarboxylic acid. Nitric acid is able to oxidize the terminal groups of
aldoses, but leaves the secondary hydroxyl groups unchanged. The dicarboxylic
acid formed from galactose is called mucic acid and is insoluble in cold aqueous
solution

Chemical Composition:
Aldose and dicarboxylic Acid
Positive Results:
 When galactose solution is
heated with concentrated nitric
acid, a dicarboxylic acid called
mucic acid or galactaric acid is
formed as white precipitate.

C. POLYSACCHARIDE TEST

1. Iodine Test

Procedures:

I. Place a small pinch of starch powder on a glass over a piece of white paper
II. Drop Lugol’s Solution on it.
III. Note the result
Principles:
Iodine’s test is for the presence of starch in which the sample turns into blue-
black in color when a few drops of potassium iodide solution is placed on sample.
The reaction is due to the formation of polyiodide chains from the reaction of
starch and iodine.

Chemical Composition:
Starch powder and Lugol’s solution
Positive Results:

The starch powder

quickly changes to
black color mainly
because of the
presence of iodine.

2. Hydrolysis Test

Procedures:
I. Place 20ml of starch solution on a beaker
II. Add 2 drops of concentrated HCL
III. Boil
IV. At intervals of 3mins, test a drop with Lugol’s and a small proportion of
Benedict’s reagent. Until Iodine test becomes negative and the benedict’s
test becomes positive,
 Principles:

Hydrolysis test is used to identify bacteria that can hydrolyze starch (amylose
and amylopectin) using the enzymes a-amylase and oligo-1, 6-glucosidase. The
iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the
starch will create a clear zone around bacterial growth

Chemical Composition:
Starch and Concentrated HCL
Positive Results:

A transparent clear zone is formed

around the colonies that hydrolyze
starch while the rest of the plate
show a blue coloration as iodine
forms the colored complex with
starch

II. QUESTIONS

1. Approximately how many times faster does fructose react to give a colored
product compared with the other sugar samples?
 Fructose is a ketose sugar that will react faster with the reagent to form the
cherry-red complex after 1 minute. Compared to the other sugars which
reacts at a slower rate

2. How important is time in the characterization of the sugar using Barfoed’s test?
 Since the Barfoed’s test is used for distinguishing monosaccharides from
reducing disaccharides. Monosaccharides usually react in about 1-2 minute
while the reducing disaccharides take 7-12 minutes to react with the reagent.
Brick red colour is obtained in this test which is due to cuprous oxide

3. Differentiate Benedict’s from Fehling’s test?

 The difference is that the Fehling’s test solution changes its colour but at the
end of the boiling process, it turns back to its original colour with red ppt. The
Benedict’s test changes color totally until the end of the procedure.

4. What are the differences between Benedict’s and Fehling’s tests? Which one
is more sensitive?
  The Benedict’s test is more sensitive because it undergoes to a series of
change in color such as blue, green. Yellow and orange while exposed to
heat.

5. What precipitate results when reducing sugars are heated with Fehling’s,
Benedict’s and Barfoed’s reagent?
 Fehling's solution contains blue alkaline cupric hydroxide solution, When
heated with reducing sugars it gets reduced to yellow or red cuprous oxide
and is precipitated. Hence, formation of the yellow or brownish-red colored
precipitate helps in the detection of reducing sugars in the test solution. In
the Benedict's solution it is cognised of copper sulphate solution, which
gives a blue colouration. This reacts with the electrons from the ketone or
aldehyde group of the free reactive carbonyl group on the carbohydrate to
form cuprous oxide which is a red-brown precipitate. The color of
the precipitate is dependent on the volume of reducing present. This
means that the snore free carbonyl groups present, the increase in the
amount of precipitate formed. On the other hand, the Barfoed’s reagent
comprises of cupric acetate and acetic acid in solution. Monosaccharides
readily react with the reagent to causes a reduction and this is because
monosaccharides are oxidized readily in weak acid solutions.
Disaccharides also can reduce the cupric ions; however, this reaction is
much slower. This will result in a red-brown colouration formed within the
solution. The monosaccharides then reduce the phosphomolybdic acid in
the phosphomolybdate colour reagent to form phosphomolybdenum blue.
Disaccharides also cause this reduction, however the reaction may be
slower or none existent since it is dependent on the reducing nature of the
sugar. Thus, the blue formed causes a deep blue colouration in the
solution in the monosaccharide solution.

6. Complete the table. For positive results draw a test tube and color the positive
results

Test Reagent Tests for Results (Positive)

When a piece of starch
powder was placed on
Iodine test
white paper and a drop of
Lugol’s solution was added Presence of starch.
the starch changed its color
to blue-black.
General test for all
carbohydrates or any
When a carbohydrate is
compound containing a
treated with mineral acid.
Molisch’s test carbohydrate residue in
Molisch’s reagent 5% a
molecule
napthol in alcohol
 When a solution of reducing
sugar is heated with an
Polymerizes to form
alkali (NaOH), it turns
resinous substance
Moore’s test yellow to orange and finally
called caramelization
dark brown, liberating the
odour of caramel
When benedict’s reagent
was added to glucose
Benedict’s test A test for reducing
solution it changed its color
sugar.
to green yellow orange red
and brick red
Barfoed’s reagent
comprises of cupric acetate
Used to distinguish
and acetic acid in solution.
monosaccharides from
Barfoed’s test This will result to a
disaccharides
phosphomolybdenum blue.

When Fehling’s A is blue

aqueous solution of copper
II sulphate pent hydrate
Fehling’s test crystals, while Fehling’s B is Fehling’s test is used in
clear solution of aqueous detecting the presence
potassium sodium tartrate of aldehyde but not

(Rochelle salt) and a strong ketones

alkali (sodium hydroxide)
formed a brownish-red
colour.
When Nylander's

reagent with I ml of Nylander's test is

Nylander’s test 5% glucose used to detect the

originally a clear presence of reducing

solution and changed sugars (glucose or

into dark brown or (Fructose).

Black color.
 Seliwanoff's reagent

contains a nom Seliwanoff's test is

Seliwanoff’s test oxidizing acid such as used to distinguish

HCL or resorcinol ketoses from

then reacts to a aldehydes in

furfural derivatives to

form a cherry-red

Complex.
When 1ml of 5%

glucose solution was Picric Acid test is a

Picric Acid test added of 0.5 ml of test for reducing

saturated picric acid sugars to form a red

and Na2C03 the colored picramic

color was light yellow Acid.

to dark yellow and to red

color
When galactose Mucic Acid test is

solution heated used to identify the

Mucic Acid test with concentration presence of the sugar

nitric acid, a Galactose.

dicarboxylic acid

called mucic acid or

galactaric acid is

formed as a white

ppt.

7. Based on Oxidation

Sugar Solution Mucic Acid Fehling’s Benedict’s Barfoed’s

Test Test Test
(Description)
Glucose - + + +
 Fructose - + + +
Galactose + + + +
Maltose - + + -
Sucrose - - - -
Lactose + + + -

III. CONCLUSION

In this activity, this will show all the different types of tests of the carbohydrates
including the monosaccharides, disaccharides and polysaccharides using the different tests. In
monosaccharides and disaccharides there are two tests these are: Moore's test which is a test
that will change the color of the solution into a caramel or a caramelization process. While, in
Molisch's test is a test for all carbohydrates and will form a furfural derivatives.

On the other hand, under the Reduction test we have 8 kinds of tests. The first test was the
Fehling's test where it produces a brownish red precipitate. The Benedict's test will change its
original color to green, yellow, orange, red and brick red. The Nylander's test have a clear
solution which will changed its color to yellow, orange and finally to dark brown. The Barfoed’s
test will obtained a brick red color due to formation of cuprous oxide. The Picric Acid test will
then form a red color picramic acid. The Seliwanoff's test will produce a cherry-red complex
which is formed in the reaction indicating the presence of the ketose sugar. The Bail’s test will
form a green yellow color change in the solution.

The Mucic Acid test or galactaric acid will form a white precipitate which will be the positive
result of the solution. In the polysaccharides consists of Iodine test and Hydrolysis test. The
Iodine test will make the starch stain into a blue-black in color since amolvsis is present while,
the Hydrolysis test is performed on disaccharides and polysaccharides to breakdown to the
monomer monosaccharide units. This test will enable the breakdown of sucrose into fructose
and glucose that will then form a clear solution. So, as you have seen above those with colored
tubes are the positive results of each test that was performed. In this activity, we will able to
understand the principles, procedures or preparation, chemical composition and of course, their
respective results of all the carbohydrates