* to learn techniques, understand principles in

glycogen isolation
* to explain the principle behind using cold
precipitation for isolating glycogen
* to confirm presence of carbohydrates using
qualitative tests
 Carbohydrates are the most abundant
- biomolecules in nature.

n n

This formula is only true for simple

sugars or monosaccharides. They are the
Made up of poly-hydroxy monomeric units of oligosaccharides and
aldehydes or ketones bonded polysaccharides.
together by glycosidic bonds.
Glycogen is a polysaccharide that serves
as the fuel storage form of glucose in
animal and fungal cells.

* appears as granules in cells

* soluble in water
* insoluble in alcohol
The source of glycogen used in the
experiment is chicken liver. Glycogen is
mainly stored in the liver and muscles. It
can undergo chemical hydrolysis or
enzymatic hydrolysis.
Glycogen is soluble in water due to its
structure wherein the hydrophilic
hydroxyl groups are allowed to interact
with water. It is insoluble in alcohol,
however, and promotes precipitation
instead. Low temperature helps in the
completion of the reaction.
Chemical hydrolysis is achieved through
the addition of a strong acid such as HCl.
The glycosidic bonds are broken.
Monomeric units of glycogen, which is
glucose, are produced.
Table 1. Qualitative results for the Molisch test.

(+) 1% (-) distilled

crude NH
glucose H2O

no
purple ring purple ring* purple ring*
discoloration

Molisch test is a general test that used to detect the presence of

carbohydrates. It is based on the hydrolysis of the carbohydrate to
its monomeric unit. In the presence of a strong acid (HCl, H2SO4),
dehydration occurs and produces a compound called furfurol
(furfural derivative) and forms a violet ring or coloration.
The Molisch test is non-specific for
carbohydrates, as well as nucleic acids
that contain pentoses, and even
combined carbohydrates such as
glycolipids and glycoproteins. The
hydrolysis of the carbohydrates present
in strong acid produces different
furfural derivatives that give positive
results (purple ring).
Table 2. Qualitative results for the Benedicts test.

(+) 1% (-) distilled

crude NH
glucose H2O

blue blue red* blue*

Benedicts test is a specific and sensitive colorimetric method in

determining the presence of reducing sugars. High concentration
of reducing sugar is indicated by the formation of a brick-red
precipitate. Blue indicates negative results.
Reducing sugars are capable of acting
as reducing agents due to the presence
of a free aldehyde or a ketone group. All
monosaccharides are reducing sugars
(aldoses, ketoses). Some disaccharides,
oligosaccharides, and polysaccharides
can be reducing sugars in the presence
of a reducing end, that is the
monomeric end that has a free anomeric
carbon.
The Benedicts reagent is Cu2+ ions in
aqueous sodium citrate. The following
reaction occurs in the presence of a
reducing sugar:

Na2CO3 + 2H2O 2NaOH + H2CO3

2NaOH + CuSO4 Cu(OH)2 + Na2SO4
Cu(OH)2 + Na3C6H5O7 Cu(OH)2:Na3C6H5O7complex
Cu(OH)2 CuO + H2O

D-glucose + 2CuO D-gluconic acid + Cu2O
 none

Sodium carbonate is responsible for the

alkalinity. Cupric (II) ions are provided 0.1-0.5
by the copper sulfate. Sodium citrate,
which is a chelating compound, ensures
the retention of the metallic ions in the 0.5-1.0
solution. The reduction of copper (II)
oxide by the reducing sugar produces
the brick-red precipitate [copper (I) 1.5-2.0
oxide].

>2.0
Table 3. Qualitative results for the Osazone test.

(+) 1% (-) distilled

crude NH
glucose H2O

yellow yellow yellow*

clear
4:30 minutes* 1:15 minutes* 4:00-5:00 minutes*

Osazone test is used to detect the presence of reducing sugars due

to the formation of osazone (based on time). Sugar osazones are
yellow, crystalline compounds characteristic to the carbohydrate.
The time for the crystals to form is also dependent on the type of
sugar present in the sample.
Phenylhydrazine is used for this test.
Formation of phenylhydrazone is due to
the reaction of the compound with the
carbonyl group of the reducing sugar in
high temperature. This then reacts with
two molecules of phenylhydrazine
producing the osazone crystals.
Fructose and mannose are both
expected to form analogously to
glucosazone crystals. Phenylhydrazine
reaction with hexoses only involves the
C1 and C2 positions. The difference in
configuration in these positions does not
affect the shape of the crystals formed,
which are needle-shaped.
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