Carbohydrates

Introduction
Carbohydrates are polymers of sugar molecules that serve as storage molecules or as
structural molecules. Starch and glycogen are examples of storage molecules. Hydrolysis of
either starch or glycogen releases the monosaccharide glucose, which may then serve as fuel for
cellular work or as a carbon source for synthesis of other organic molecules. Cellulose, which
makes plant cell walls tough, and chitin, the main ingredient of an insects exoskeleton, are
examples of structural molecules.
Carbohydrates classified as simple or complex. Simple carbohydrates, often called
monosaccharides or simple sugars, cannot be broken down into smaller carbohydrate molecules.
Complex carbohydrates can be broken down into smaller carbohydrate units through a process
known as hydrolysis.
Carbohydrates as the name indicate are made from carbon (C) and hydrate (H2O) and
was originally applied to the compounds that gave the (CH2O)n formula (Applicable for
monosaccharides and for example C6H12O6) and often referred to as CHO. Disaccharides are
formed through the condensation (Polymreraization) reaction to remove one H2O molecule
and the formation of glycosidic bond between the two monosaccharides, disaccharides usually
have C2nH2n-2On-1 formula and for example C12H22O11. Polysaccharides form through the
addition of monosaccharides and the removal of one H2O moleculkes per each addition of the
monosaccharides. Polysaccharides usually have (CnH2n-2On-1)y formula, this formula represent a
long monosaccharides residues minus H2O.
Chemically, carbohydrates are either aldehydes or ketones derivatives of polyhydroxylic
compound that known as aldoses and ketoses. Carbonyl group give aldoses and ketoses the
reducing properties. Sucrose is the most common non-reducing sugar as the reducing carbonyl
groups of glucose (C1) and fructose (C2) are involved in the glycosidic bond.

Monosaccharides:

Arabinose

Fructose

Glucose

Galactose

Mannose

Biochemistry laboratory notes / Carbohydrates by Amjad I.A. Hussein

Ribose

Xylose
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Disaccharides:

Sucrose

Sucrose Haworth's Representation

Lactose

Maltose

Polysaccharides:

Starch

Cellulose
Exp. # 1 Molischs test
Principle:
Concentrated sulphuric acid hydrolyses glycosidic bonds to give the monosaccharides,
which are then dehydrated to furfural and its derivatives. These products then combine with
sulphonated naphthol to give a purple complex.

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This reaction is a general one for the presence of all carbohydrates (Monosaccharides
give a rapid positive test. Disaccharides and polysaccharides react slower) and other organic
compounds that give furfural with concentrated sulphuric acid.
Materials and reagents:
1. Carbohydrate solutions (1 g/l and 10 g/l).
2. Sulphuric acid (concentrated).
3. Molischs reagent (-naphthol 50 g/l in ethanol, prepare fresh).
Method:
1- Add two drops of the Molischs reagent to 2ml of test solution.
2- Carefully pour about 1ml of conc. H2SO4 down the side of the tube so as to form two layers.
3- Observe any colour change at the junction of the two liquids. The formation of a purple
product at the interface of the two layers indicates positive result.

Negative result (left)

Positive result (right)

4- If the purple layer is difficult to see, mix slowly and carefully (large amounts of heat is
generated) and observe the colour. If the test solution sugar concentration high enough all the
solution become purple color.

Negative result (left)

Positive result (right)

Exp. #2 Anthrone test

Principle:
The anthrone reaction is another general test for carbohydrates. The principle is the same as
that in Molischs test except that the furfural reacts with anthrone to give a blue-green complex.
There is a linear relationship between the color (absorbance) and the amount of sugar that was
present in the original sample. The absorbance of the resulted colored solution can be measured
at 620 nm.
Materials and reagents:
1. Carbohydrate solutions (1 g/l and 10 g/l).
2. Anthrone solution (2g/L in conc. H2SO4).
Method:
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1- Add five drops of the test solution to about 2 ml of the anthrone reagent.
2- Mix and observe the colour change.

Reaction of reducing sugar

Reducing sugars are those sugars capable of reducing copper ions in a solution to cuprous
oxide. The reducing sugars include:
(1) All of the simple sugars (monosaccharides).
(2) A few of the disaccharides (including lactose and maltose, but not sucrose).
(3) None of the polysaccharides.
Background: If a suspension of copper hydroxide in alkaline solution is heated, then
black cupric oxide is formed.
Cu(OH)2 CuO + H2O
However, if a reducing substance is present, then rust-brown cuprous oxide is precipitated.
2Cu(OH)2 Cu2O + H2O + O2
In practice, an alkaline solution of a copper salt and an organic compound containing
alcoholic OH is used rather than the above suspension. Under these conditions, the copper
forms a soluble complex and the reagent is stable.
Carbohydrates with a free or potentially free aldehyde or ketone groups have reducing
properties in alkaline solution. In addition, monosaccharides act as reducing agents in weakly
acidic solution.

Exp. #3 Benedicts test

Principle:
Reducing sugars are oxidized by the copper ions in the Benedicts solution to form a
carboxylic acid and a reddish precipitate of cuprous oxide within three minutes in alkaline
medium. Benedict is a more sensitive alternative modified reagent to the original Fehling's test
to produce a single solution which is more convenient for tests, as well as being more stable than
Fehling`s reagent.

Aldehyde (Carbonyl gp.)

Benedicts solution

Carboxylic acid

Reddish precipitate

Materials and reagents:

1. Carbohydrate solutions (1 g/l and 10 g/l).
2. Benedicts reagent. (To prepare one litre of Benedicts reagent: mix 173 g of tri-sodium citrate
and 100 g of sodium carbonate with 800 ml of distilled water. Warm to dissolve, then cool and
filter, add more distilled water to make the volume to 850 ml. Dissolve 17.3 g of copper sulphate
in 100 ml distilled water and stir slowly into the first solution. Bring the volume to 1 litre with
distilled water).
Method:
1- Add five drops of the test solution to 2 ml of Benedicts reagent and place in a boiling water
bath for 5 min.
2- Examine the sensitivity of benedicts reagent using increasing dilutions of glucose.
3- The formation of a orange-brown precipitate within 3-5 minutes indicates positive result.
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Negative result (left)

Positive result (right)

Exp. #4 Barfoed's test

Principle:
Barfoed's test is similar to Benedicts test in using cupper ions as an oxidising agent to
form a carboxylic acid and a reddish precipitate of cuprous oxide within three minutes.
However, the test medium is weakly acidic, therefore, only reducing monosaccharides give
positive result using Barfoed's reagent. Prolonged boiling may hydrolyse disaccharides (slow
reacting) to give a false positive reaction. The precipitate of cuprous oxide is less dense than
Benedicts test, therefore, its recommended to leave the tube to stand to allow the precipitate to
settle. The colour of the cuprous oxide is also different being a brick-red rather than orangebrown in Benedicts test.

Materials and reagents:

1. Carbohydrate solutions (1 g/l and 10 g/l).
2. Barfoeds reagent (Dissolve 13.3 g of copper acetate in 200 ml water and add 1.8 ml of glacial
acetic acid).
Method:
1- Add 1 ml of the test solution to 2 ml of Barfoeds reagent.
2- Boil for 1-3 min and allow to stand.

Negative result (left)

Positive result (right)

2- The formation of a brick-red precipitate within 3-5 minutes indicates positive result.

Exp. #5 Osazones preparation

Principle:
Phenylhydrazine react with the carbonyl group of the reducing sugar to give
phenylhydrazone, which then reacts with a further two molecules of phenylhydrazine to form
osazone (Two molecules of phenylhydrazine condense with each molecule of reducing sugar).
Monosaccharides rapidly form yellow solid osazones. Mannose forms white crystals.
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Disaccharides Sucrose does not react to form osazone for at least 30 minutes when it begins to
hydrolyze.

Note the time required for the formation of the osazones, which can be a valuable aid in
distinguishing among various sugars. The following figures are the times required for the
osazone to precipitate from the hot solution: fructose in 2 min; glucose in 4-5 min; mannose in
4 min (white precipitate at room temperature, but the yellow osazone separates on heating);
xylose in 7 min; arabinose in 10-15 min; galactose in 15-20 min. Sucrose and other
disaccharides in at least 30 min (owing to hydrolysis and formation of glucosazone).
Materials and reagents:
1. Carbohydrate solutions (1 g/l and 10 g/l).
2. Acetic acid (glacial).
3. Phenylhydrazine solution.
4. Sodium acetate.
5. Microscope.
Method:
1- Acidify 5 ml of the sugar solution with 10 drops of glacial acetic acid.
2- Add 3 drops of phenylhydrazine solution.
3- Add a good knife point of solid sodium acetate.
4- Place the test tube in a beaker of boiling water.
Optional step:
- With occasional shaking, filter the solution after 5 min.
- Then boil for a further 20 min.
5- Note the time of the beginning of precipitation.
6- After 25 min, remove the test tube from the hot water bath and set it a side to cool.
7- A small amount of the liquid and solid is poured on a glass slide. Tip the watch glass from
side to side to spread out the crystals, and absorb some of the liquid with a piece of filter paper
(taking care not to crush or break up the clumps of crystals).
8- Examine the crystals under a low-power microscope and compare with photomicrographs.
__The formation of tarry products due to oxidation of the phenylhydrazine may be prevented by
the addition of 0.5 mL of saturated sodium bisulfite solution. This should be done before
heating if it is desired to isolate the osazone and determine its melting point.

Glucosazone

Maltosazone

Lactosazone

Tests for individual carbohydrates

Exp. #6 Bial's (Orcinol) test
Principle:
This test is specific for pentoses. The concentrated HCl in Bial's reagent dehydrates
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pentoses to form furfural. Furfural further reacts with orcinol and the iron ion to produce a bluish
(Blue-green) product extractable into amyl alcohol. The test is used for the determination of
RNA and the detection of pentosuria. The reaction is not absolutely specific for pentoses since
prolonged heating of some hexoses yields hydroxy-methyl furfural which also reacts with orcinol
to give colored complexes.
Materials and reagents:
1. Carbohydrate solutions (1 g/l and 10 g/l).
2. Bials oricinol reagent (Dissolve 1.5 g of oricinol in 500 ml of conc. HCl and add 20 drops of
100 g/l solution of FeCl3).
3. Amyl alcohol.
Method:
1- Add 1 ml of the test solution to 2.5 ml of Bials reagent.
2- Heat until boiling commences.

Negative results (left & middle)

Positive result (right)

2- The formation of a blue-green color indicates a positive result for pentoses.

3- Note that Hexoses generally react to form green, red, or brown products.
4- Cool the tube, then add 2-3 ml of amyl alcohol and shake.

Exp. #8 Seliwanoffs test

Seliwanoffs test distinguishes between aldose and ketose sugars. The test is based on
the fact that, when heated, ketoses are more rapidly dehydrated than aldoses to give furfural
derivatives. The reagents consist of resorcinol and hydrochloric acid; 1) the acid hydrolysis
polysaccharides and oligosaccharides yields simpler sugars; 2) Then the dehydrated ketose reacts
with the resorcinol to produce a deep cherry red color. Aldoses may react slightly and more
slowly to produce a faint pink color. Fructose and sucrose (A disaccharide consisting of fructose
and glucose) are two common sugars that give a positive test.

Resorcinol

Materials and reagents:

1. Carbohydrate solutions (1 g/l and 10 g/l).
2. Seliwanoff's reagent (Dissolve 0.5 g of resorcinol in 1000 ml of 3M HCl).
Method:
1- Add 2 drops of the test solution to 2.5 ml of Seliwanoff's reagent.
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2- Warm in boiling water for 1-2 min.

Negative result (left)

Positive result (right)
3- The formation of a red color within 2 min indicates a positive result for ketoses (Fructose and
Sucrose).
4- Note that prolonged heating with aldoses generally react to form faint pink color product.

Exp. #9 Tests for sucrose

Principle:
Sucrose is the only common non-reducing disaccharide, so that it doesnot reduce alkaline
copper solutions or form an osazone. Sucrose is therefore hydrolysed in acid solution to glucose
and fructose, which are then tested for specified tests.
Materials and reagents:
1. Sucrose solutions (1 g/l and 10 g/l).
2. Concentrated HCl
3. Sodium hydroxide (5 mol/l).
4. Benedicts, Barfoeds, Osazones and Seliwanoffs reagents.
Method:
1. Add 5 drops of conc. HCl to 5 ml of sucrose solution.
2. Heat for 5 min on a boiling water bath.
3. Cool down then add NaOH to give a neutral or slightly alkaline solution.
4. Perform reduction tests and Seliwanoffs test on the hydrolysed solution.
5. Prepare the Osazones using the hydrolysed solution.

Exp. #10 Iodine test

Principle:
Iodine complexes with polysaccharides. Starch form a blue-black product, while
glycogen and partially hydrolysed starch give red-brown color. As some of the polysaccharides
are not water soluble a suspension or a solid can be directly tested by placing the iodine solution
directly on the solid.
Materials and reagents:
1. Carbohydrate solutions (1 g/l and 10 g/l).
2. Iodine (IKI) solution (5 mmol/l in KI 30 g/l).
Method:
1- Acidify the test solution with dilute HCl.
2- Add 2 drops of iodine solution.
3- Compare the colors obtained from different solutions, starch give a blue-black complex.

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Negative result (left)

Positive result (right)

Exp. #11 The hydrolysis of polysaccharides

Principle:
Polysaccharides contain only one reducing group for several hundreds or more residues
so that they are effectively non-reducing. Acid hydrolysis gives the constituent monosaccharides,
which are then tested for specified tests.
Materials and reagents:
1. Cellulose, glycogen, starch and inulin solutions (10 g/l).
2. Concentrated HCl
3. Sodium hydroxide (5 mol/l).
4. Iodine (IKI) solution (5 mmol/l in KI 30 g/l).
5. Benedicts, Osazones and Seliwanoffs reagents.
Method:
1. Add 10 drops of conc. HCl to 10 ml of polysaccharide solution.
2. Boil gently.
3. Remove one drop of the solution with a Pasteur pipette every minute and mix with one
drop of iodine on a white tile.
4. At the same time, remove three drops of the solution, neutralize with NaOH, and add to 5
ml of Benedicts solution.
Continue removing samples up to 6 min or when appropriate.
Place all the tubes containg Benedicts solution in boiling water bath for 3 -5 min and
allow to cool.
Explain the results.
5. Neutralize the remaining solution with alkali and identify the sugars present as far as possible.

Carbohydrates concentration
Many different methods have been used to determine the carbohydrates concentration.
Commonly used methods include polarimetry and refractive index that rely on the change in
some physicochemical characteristic as the carbohydrate concentration varies.

Polarimetry (Polarimeter):
Molecules that contain an asymmetric carbon atom have the ability to rotate plane
polarized light. A polarimeter is a device that measures the angle that plane polarized light is
rotated on passing through a solution. A polarimeter consists of a source of monochromatic light,
a polarizer, a sample cell of known length, and an analyzer to measure the angle of rotation. In
the following graph is an ordinary optical polarimeter.

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The extent of polarization is related to the concentration of the optically active molecules in
solution by the equation,
a=[a]lc,
where,
- a is the measured angle of rotation,
- [a] is the optical activity (which is a constant for each type of molecule),
- l is the path-length and c is the concentration.
The overall angle of rotation depends on the temperature and wavelength of light used
and so these parameters are usually standardized to 20oC and 589.3 nm (the D-line for sodium).
A calibration curve of a versus concentration is prepared using a series of solutions with known
concentration, or the value of [a] is taken from the literature if the type of carbohydrates present
is known. The concentration of carbohydrates in an unknown sample is then determined by
measuring its angle of rotation and comparing it with the calibration curve.

Refractive Index (Refractometer):

The refractive index (n) of a material is the velocity of light in a vacuum divided by the
velocity of light in the material (n = c/cm). The refractive index of a material can be determined
by measuring the angle of refraction (r) and angle of incidence (i) at a boundary between it and
another material of known refractive index (Snells Law: sin(i)/sin(r) = n2/n1). In practice, the
refractive index of carbohydrate solutions in the refractometer is usually measured at a
boundary with quartz. The refractive index of a carbohydrate solution increases with increasing
concentration and so can be used to measure the amount of carbohydrate present.
The RI is temperature and wavelength dependent and so measurements are usually
made at a specific temperature (20 oC) and wavelength (589.3nm). This method is quick and
simple to carry out and can be performed with simple hand-held instruments. It is used routinely
in industry to determine sugar concentrations of syrups, honey, molasses, tomato products and
jams.

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Scheme for the identification of an unknown

The following is a postulated scheme for the identification of a carbohydrate solution.
However, this identification is presumptive and need a further confirmative test like paper or
thin layer chromatography.

Molischs test
Carbohydrates
Positive
+

Benedicts test

Barfoeds test

Bials test
Positive

Negative

Lactose
Maltose

Iodine test
Blue-Black
Starch

Brown

Negative

Glycogen

Inulin
Sucrose

Negative
Seliwanoffs test

Osazones preparation
Ribose
Arabinose
Xylose

Seliwanoffs test

Positive

Positive
Fructose

Negative

Sucrose

Osazones preparation
4 min
Glucose

20 min
Galactose

Biochemistry laboratory notes / Carbohydrates by Amjad I.A. Hussein

Room temp.
Mannose

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