Chemical Tests for
Carbohydrates
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Carbohydrate
Carbohydrates are the key source of energy used by living things.
Also serve as extracellular structural elements as in cell wall of bacteria and plant.
Carbohydrates are defined as the polyhydroxy aldehydes or polyhydroxy ketones.
Most , but not all carbohydrate have a formula (CH2O)n (hence the name hydrate of
carbon)
In human body, the D-glucose is used.
Simple sugars ends with –ose.
Several classifications of carbohydrates have
proven useful, and are outlined in the following
table
Classification
1-Simple sugar (one unit) :
Monosaccharides contain one monosaccharide unit.
2-Complex sugar (more than one) :
•Disaccharides contain two monosaccharide units.
•Oligosaccharides contain 3-9 monosaccharide units.
•Polysaccharides can contain more than 9 monosaccharide units.
-Complex carbohydrates can be broken down into smaller sugar units
through a process known as hydrolysis.
Reducing and non reducing sugars
Reducing and non reducing sugar :If the oxygen on the anomeric carbon
of a sugar is not attached to any other structure, that sugar can act as a
reducing agent and is termed a reducing sugar.
Solubility of sugars [physical
property]
Monosaccharide and disaccharide can be dissolved freely in water because
water is a polar substance, while polysaccharide cannot be dissolved easily
in water, because, it has high molecular weight , which give colloidal
solutions in water.
Chemical Properties of
:Carbohydrates
Molisch Test: specific for carbohydrates.
Iodine Test: specific for polysacgarides.
Benedict's Test: presence of reducing sugars.
Barfoed's Test: test used for detecting the
presence of monosaccharides.
Bial's Test: used to detect pentose [5C]
monosacharides.
Seliwanoff's Test: distinguish between aldoses
and ketoses.
Molisch test
This test is specific for all carbohydrates Monosaccharide gives a rapid
positive test, Disaccharides and polysaccharides react slower.
Objective: To identify the carbohydrate from other macromolecules,
lipids and proteins.
Principle: The test reagent(H2SO4) dehydrates pentose to form furfural
and dehydrates hexoses to form 5- hydroxymethyl furfural. The furfural
and 5- hydroxymethyl furfural further react with α-naphthol present in
the test reagent to produce a purple ring.
Method :
1-Two ml of a sample solution is placed in a test tube.
2-Two drops of the Molisch reagent (which α-napthol in 95% ethanol) is added.
3-The solution is then poured slowly into a tube containing two ml of concentrated
sulfuric acid so that two layers form, producing violet ring appear as liaison
between the surface separations
Interpretation:
This is a sensitive but a non-specific test is given positive by all types of
carbohydrates. If oligosaccharides or polysaccharides are present, they are first
hydrolysed to mono saccharides which are then dehydrated to give the test
positive.
An appearance of reddish violet or purple coloured ring at the junction of two
liquids is observed.
Iodine Test
Principle: Iodine forms a coordinate complex between the helically coiled
polysaccharide chain and iodine cantrally located within the helix due to
adsorption. The colour combined depends upon the lenghth of the
unbranched or linear chain available for complexformation
Method :
You will test glucose, fructose, lactose, sucrose, starch, water, and your
unknown.
Add 1 mL of the solution to be tested to each of 7 labeled test tubes.
Add 3 drops of iodine solution to each of the 7 test tubes, and mix each
tube. Compare the colors and record your observations
Interpretation: This is a test for polysaccharides which is indicated by
formation of blue/brown/red colour. No change in colour is indicative of
presence of mono or disaccharide.
Benedict's test
Objective: To detect the presence of reducing sugars.
•All monosaccharides are reducing sugars; they all have a free reactive
carbonyl group.
•Some disaccharides have exposed carbonyl groups and are also reducing
sugars. Other disaccharides such as sucrose are non-reducing sugars and
will not react with Benedict's solution
-Large polymers of glucose, such as starch, are not reducing sugars, since
the concentration of hemiacetal groups is very low.
Principle : Carbohydrates with free aldehyde or ketone groups have
the ability to reduce solutions of various metallic ions.
Reducing sugars under alkaline conditions tautomerise and form
enediols. Enediols are powerful reducing agents . They reduce
cupric ions (Cu3+) to cuprous (Cu2+) form and are themselves
converted
to sugar acids, The cuprous ions combine with OH- ions to form
yellow cuprous hydroxide which upon heating is converted to red
cuprous oxide.
Method
• One ml of a sample solution is placed in a test tube.
• Two ml of Benedict's reagent is added.
• The solution is then heated in a boiling water bath for five
minutes. A positive test is indicated by: The formation of a
reddish precipitate.
Interpretation: It is semi quantitative test. The colour of the
precipitate gives a rough
estimate of a reducing sugar present in the sample: Blue: none,
Green : up to 0.5gm% (+), Green precipitates : 0.5-1.0gm% (++),
Yellow precipitates : 1.0-1.5gm% (+++), Orange precipitates : 1.5-
2.0gm% (++++), Brick red precipitates : >2.0gm% (+++++)
Barfoed’s Test
Objective : To distinguish between mono- , di- and poly saccharides.
Principle : Barfoed’s test used copper (II) ions in a slightly acidic medium.
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.
-The nonreducing sugars give negative result.
Method
Place one ml of a sample solution in a test tube.
Add 3 ml of Barfoed's reagent (a solution of cupric acetate and acetic acid).
boil it for one minute in the water bath allow to stand for a few minutes. Formation of a
red precipitate of cuprous oxide in the bottom and along the sides of the test tube
immediately, only monosaccharides answer this test. Since Barfoed’s reagent is slightly
acidic, This test is specific for monosaccharides.
Bial’s Test
Objective: To distinguish between pentose
monosaccharide and hexose monosaccharide
Principle: Bial’s test uses concentrated HCl as a
dehydrating acid and orcinol + traces of ferric chloride
[FeCl3] as condensation reagent. The test reagent
dehydrates pentoses to form furfural. Furfural further
reacts with orcinol and the iron ion present in the test
reagent to produce a bluish or green product, while
hexoses yield muddy-brown to grey condensation
product.
Method :
Put 2 ml of a sample solution in a test tube.
Add 3 ml of Bial's reagent (a solution of orcinol, HCl and ferric chloride) to each
tube.
Heat the tubes gently in hot water bath.
If the color is not obvious, more water can be added to the tube
Interpretation:
This test is specific for pentoses indicated by formation of bluish product. All other
colors
indicate negative result for pentoses. Hexoses generally react to form green,red
or brown
products.
Seliwanoff's Test
Objective:
used to distinguish between aldoses (like glucose) and
ketoses (like fructose).
Principle:
Seliwanoff's Test uses HCl as dehydrating agent and
resoncinol as condensation reagent. The test reagent
dehydrates ketohexoses to form 5-
hydroxymethylfurfural. 5-hydroxymethylfurfural further
condenses with resorcinol present in the test reagent to
produce a cherry red product within two minutes.
-Aldohexoses react to form the same product, but do so
Method
One half ml of a sample solution is placed in a test tube.
Two ml of Seliwanoff's reagent (a solution of resorcinol and HCl) is added.
The solution is then heated in a boiling water bath for two minutes.
Interpretation:
This test is given positive by ketohexoses so it is answered by fructose,
sucrose and
other fructose containing carbohydrates.
This test distinguishes between glucose and fructose.
Overheating of the solution should be avoided. Upon continuous boiling,
aldoses get converted to ketoses and give a positive reaction with seliwanoff
reagent
