Biochemistry II :( Lab.

6)
By: Asmaa Gebril

Qualitative Analysis of
Carbohydrates

10 ‫ﻣﮭﻢ ﻓﻲ اﻻﻣﺘﺤﺎن وﻻ ﯾﻘﻞ ﻋﻦ‬

Carbohydrates play many important roles in biological systems. They represent the
major form of chemical energy for both plants and animals. In plants they represent
the end product of photosynthesis, and therefore connect all living systems to the
sun’s sustaining light energy. Our discussion of carbohydrates will also introduce us
to biopolymers, of whichproteins and nucleic acids also belong. One of these
polymers, the structural polysaccharide cellulose, ties more of the earth’s organic
carbonthan any other molecule.
Polymers are large molecules that are made by stringing together, likebeads on a
string, smaller units called monomers.
Poly- is the Greek prefix meaning “many”.
The names of may polymers describe what they are made from
Polyethylene is made by stringing together many ethylene units.Ethylene
(ethane) is the monomer
Polypropylene is made by stringing together many propylene units.Propylene
(propene) is the monomer.

Polysaccharides are made by stringing together many monosaccharide’s.

Monosaccharide’s (simple sugars) are the monomers.

2- Disaccharides:

3- Polysaccharides:
 Test
 The Molisch-Test:-
Principle:
Concentrated sulphuric acid hydrolyses glycosidic bonds to give the monosaccharaides, which are then
dehydrated to furfural and its derivatives. These products then combine with sulphonated naphthol togive a
purple complex.

(Disaccharides and polysaccharides react slower but Monosaccharides give a rapid positive test)

Procedure: -
1. Add 2 drops of Molisch reagent to 2 ml of the sugar solution to be
tested and mix.
2. Incline the tube, and GENTLY add 2 ml of concentrated sulfuric
acid down the side of the test tube.
3. A purple color at the interface between the sugar and acid indicates a positive result.
(Monosaccharide’s give a rapid positive test)

Barfoed's -Test:-
This test is used to distinguish between monosaccharide and disaccharides. Barfoed's test is similar to Benedict’s
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 monosaccharide’s give
positive result using Barfoed's reagent.

Preparing Reagent :
Barfoed's reagent consists of a 0.33 molar solution of neutral copper acetate in 1% acetic acid solution. The
reagent does not keep well and it is therefore advisable tomake it up when it is actually required.
 Procedure: -
1. Add 1 ml of the 1% sugar solution to be tested to 1 ml of
Barfoed's reagent.
2. Place test tubes into a boiling water bath and heat for 2-10
minutes. Remove the tubes from the bath and allow cooling.
3. Formation of scanty red precipitate, is a positive result for
reducing monosaccharides.
4.
(Monosaccharide’s give a rapid positive test between 2-3 min;
while Disaccharide’s give a positive result after 10 min)

 Seliwanoff’s -Test:-
This test is used for identification of keto-hexoses or to
distinguish between ketoses and aldoses. Aldoses may react slightly and more slowlyto produce a faint pink
color. Fructose and sucrose (A disaccharide consisting of fructose and glucose) are two common sugars that give
a positive tes

Procedure: -
1. Add 1 mL of the 1% sugar solution to be tested to 2 mL of the reagent.
2. Heat the solution in a boiling water bath for 2 minutes.
3. A deep cherry or red color within 2 minutes indicates the
4. presence of ketohexoses.
(Sucrose gives a positive ketohexose test because of the partial hydrolysis to glucose and fructose within 5-10 min.)
  Bials -Test:
Bial’s Test is to determine the presence of pentoses (5C sugars). The components of this reagent are resorcinol,
HCl, and ferric chloride. In thistest, the pentose is dehydrated to form furfural and the solution turns bluish and
a precipitate may form. Perform this test with Arabinose, Xylose and glucose.

Procedure:-
1- Add 0.5 ml Sample to 4 ml of Bials Reagent.
2- Heat Boiling in Water Bath.
3- The Formation of a Blue-Green Color positive Result for pentose.
compounds (pentosans) will give a positive test.

Benedict's -Test:-
also called( Benedict's solution): is a chemical reagent named after an American chemist, Stanley Rossiter
Benedict Benedict's reagent is used asa test for the presence of reducing sugars. This includes all
monosaccharides and many disaccharides, including lactose and maltose. Even more generally, Benedict's
test will detect the presence of aldehydes, and alpha-hydroxy-ketones, including those that occur in certain
ketoses.Thus, although the ketose fructose is not strictly a reducing sugar, it is an alpha-hydroxy-ketone, and

gives a positive test because it is converted to the aldoses glucose and mannose by the base in the reagent.
The copper sulphate in Benedict's solution reacts with reducing sugars. Benedict's solution can be used to tell if
there is a sugar in a substance suchas glucose in starch .
Benedict's Reagent provides a quantitative test for reducing sugars along with qualitative test. The color of
the obtained precipitate gives an idea about the quantity of sugar present in the solution. A greenish precipitate
indicates about 0.5% concentration; yellow precipitate indicates 1% concentration; orange indicates 1.5% and
red indicates 2% or higher concentration. To test for the presence of monosaccharides and reducing
disaccharide sugars in food, the food sample is dissolved in water, and a small amount of Benedict's reagent is
added. During a water bath, which isusually 4–10 minutes, the solution should progress in the colors of blue
(with no glucose present), green, yellow, orange, red, and then brick red or brown (with high glucose present).A
colour change would signify the presence of glucose. The common disaccharides lactose and maltose are
directly detected by Benedict's reagent, because each contains a glucosewith a free reducing aldehyde moiety.

Sucrose (table sugar) contains two sugars (fructose and glucose) joined by their glycosidic bond in such a way
as to prevent the glucose isomerizing toaldehyde, or the fructose to alpha hydroxy-ketone form. Sucrose is thus a
non-reducing sugar which does not react with Benedict's reagent. Sucrose indirectly produces a positive result with
Benedict's reagent if heated with dilute hydrochloric acid prior to the test, although after this treatment it is no
longer sucrose. The acidic conditions and heat break the glycosidic bondin sucrose through hydrolysis. The
products of sucrose decomposition are glucose and fructose, both of which can be detected by Benedict's reagent,as
described above.

Starches do not react or react very poorly with Benedict's reagent, due to the relatively small number of reducing
sugar moieties, which occur only atthe ends of carbohydrate chains. Benedict's reagent can be used to test forthe
presence of glucose in urine. Glucose found to be present in urine is anindication of diabetes mellitus. Once a
reducing sugar is detected in urine, further tests have to be undergone in order to ascertain which sugar is present.
Only glucose is indicative of diabetes.

What is a reducing sugar?

Sugars are classified as reducing or non-reducing based on their ability toact as a reducing agent during the
Benedict's Test. A reducing agent donates electrons during a redox reaction and is itself oxidized.
The aldehyde functional group is the reducing agent in reducing sugars. Reducing sugars have either an aldehyde
functional group or have a ketonegroup - in an open chain form - which can be converted into an aldehyde.
Reducing sugars are simple sugars and include all monosaccharides and most disaccarides.Some examples of
monosaccharides are glucose,fructose and galactose.Examples of reducing disaccharides are lactose and
maltose.

Procedure:-
1. 1 Add 1 ml of the 1% sugar solution to be tested to 2 ml of
Benedict's solution
2. Place test tubes in a boiling water bath.
3. Formation of red to brick red precipitate is a positive result .
 Starch - Iodine -Test:-Starch:
Plants store glucose as the polysaccharide starch. The cereal grains (wheat, rice, corn, oats, barley) as well as
tubers such as potatoes are richin starch.

Starch can be separated into two fractions--amylose and amylopectin. Natural starches are mixtures of
amylose (10-20%) and amylopectin (80-90%).

Amylose forms a colloidal dispersion in hot water whereas amylopectin iscompletely insoluble. The structure of
amylose consists of long polymer chains of glucose units connected by an alpha acetal linkage.

Chemical Test for Starch or Iodine:

Amylose in starch is responsible for the formation of a deep blue color in the presence of iodine. The
iodine molecule slips inside of theamylose coil.

Iodine - KI Reagent: Iodine is not very soluble in water, therefore the iodine reagent is made by dissolving
iodine in water in the presence ofpotassium iodide.

Starch Test: Add Iodine-KI reagent to a solution or directly on a potato or other materials such as bread,
crackers, or flour. A blue-black color resultsif starch is present. If starch amylose is not present, then the color
will stay orange or yellow. Starch amylopectin does not give the color, nor does cellulose, nor do disaccharides
such as sucrose in sugar.

Iodine Test: When following the changes in some inorganic oxidation reduction reactions, iodine may be used
as an indicator to follow the changes of iodide ion and iodine element. Soluble starch solution is added. Only
iodine element in the presence of iodide ion will give the characteristic blue black color. Neither iodine element
alone nor iodideions alone will give the color result.
Procedure:-
1. Add 2-3 drops of Lugol's iodine solution to 1 ml of 2% sugar solution to be tested.
2. Starch gives a blue-black color as shown. Other polysaccharides (except glycogen gives brown-black) and
monosaccharides give yellow to brown-yellow color.
  Silver mirror Test (Tollen's Reagent) for Reducing Sugars

Tollen’s test is used to distinguish between aldehydes and ketones. Tollen’s reagent, a solution of silver nitrate
and ammonia oxidizes aldehydes but not ketones. The silver ions are reduced to metallic silver which forms a “silver
mirror” in the inner surface of the test tube.
Procedure:
1. Wash the tube with a 10% solution of sodium hydroxide.
2. Add 2 ml of a 5% silver nitrate solution (AgNO3) into the washed tube, then add one drop of the 10% sodium
hydroxide solution.
3. Add one drop of 28% ammonia solution (add more drops if necessary). Shake constantly. Watch and see just
when the silver oxide just dissolves, then add 1 ml of 2% sugar solution.
4. The formation of a silver mirror in the inner surface of the test tube, is a positive result and an indication of
the presence of reducing sugar.