Methodology

Extraction of Glycogen from Chicken Liver

3 g of chicken liver was weighed and placed on a petri dish. The sample was
minced using a pair of scissors. 12 mL of boiling water was poured on it and was
stirred with a glass rod. The mixture was transferred into a small beaker and was
boiled for 2 minutes to precipitate the proteins. The mixture was poured into a
mortar and was grind thoroughly until no lumps are visible. 3 mL of distilled water
was added and the mixture was transferred into the beaker. The mixture was
heated in a boiling water for 30 minutes. Water was added to keep it from drying.
Glycogen was placed on the solution during heating. 1 mL of the 0.1% acetic acid
was added to improve the precipitation of proteins. The mixture was filtered and the
obtained glycogen solution was divided into 4 portion. These were transferred into 4
separate test tubes marked "1", "2", "3", and "4."
General Tests for Polysaccharides
A. Molisch's Test
A few drops of Molisch's reagent were added into 1 mL of glycogen
solution. 2 mL of conc. H2SO4 was poured down thhe side of the tube to form a
layer. The color was observed at the junction of the two liquids.
B. I2 Reaction

A few drops of 0.01 M I2 was added into 1 mL of the sample solution. The
mixture was warmed in a water bath and was observed if there is any change in
color. The mixture was cooled and the result was noted.

Hydrolysis of Polysaccharides

A. Acid Hydrolysis

5 drops of conc. HCl was added to 5 mL of the isolate. It was covered with
marble and boiled in a water bath for 30 minutes.

B. Enzymatic Hydrolysis

10 mL of the isolated carbohydrate was placed in a beaker. 2.3 mL of

saliva was added. The solution stand at room temperature for 30 minutes.
Changes in its viscosity was noted. The solution was introduced into a dialyzing
bag overnight in a small flask filled with 50 mL distilled water. The solution was
removed and the dialyzing bag was discarded. The solution was concentrated to
a volume of 10 mL inside the flask using an open fire.

Qualitative Tests for Carbohydrates

A. Benedict's, Barfoed's, Seliwanoff's, and Bial's Orcinol Tests

 In separate test tubes, 5 drops of the carbohydrate solution (glucose,
fructose, xylose, lactose, sucrose, and starch) and 1 mL of the required reagent
for each test were mixed. One test on the different carbohydrate solutions were
performed at the same time. All the test tubes were placed at the same time in a
boiling water bath. The tubes were removed from the water bath when the
solutions for one test produced visible results. The result and the time it took for
visible results to form in each test were noted.

B. Mucic Acid Test

3 drops of the carbohydrate solution (galactose and lactose) and 3 drops

of the conc. HNO3 were mixed on a glass slide. The mixture was passed over a
small flame until it is almost dry. The mixture was cooled at room temperature.
The crystals were examined under the microscope. The mucic acid crystals were
drawn.

C. Phenylhydrazone Test

The phenylhydrazine reagent was prepared by mixing 2 g of the

phenyhydrazine hydrochloride, 3 g of the CH 3COONa, and 10 mL of distilled
water. The reagent was placed in a warm water bath. The solution was stirred
until cleared. In different test tubes, 2 drops of the carbohydrate solution
(glucose, fructose, xylose, lactose, sucrose, and starch) were mixed with 4 drops
of freshly prepared phenylhydrazine reagent. The solutions were mixed well.
The tubes were covered with cotton. These were boiled in a boiling water bath
for 30 minutes. The time when yellow crystals first appeared were recorded. The
tubes were cooled and the crystals were observed under the microscope. The
different osazones were drawn.

Thin-layer Chromatography

40 mL of the solvent system was placed in the developing chamber. It was

covered with an inverted watch glass and was equilibrated for 10 minutes. About 2
cm from the bottom, a pencil line was lightly drawn across one end of the TLC plate.
Equidistant points were marked along the origin for the standards and the acid and
enzymatic hydrolysates. The standards were applied five time and the hydrolysates
were applied ten times using the capillary tubes. The TLC plate was placed inside
the developing chamber and was ensured that the solvent is below the line of
origin. It was covered and developed until the solvent is about 1 cm from the top of
the TLC plate. After development, the chromatoplate was removed and the solvent
front was marked with a pencil. The chromatoplate was air-dried and was sprayed
with the visualizing agent. The chromatoplate was heated on top of a hot plate. The
sugars were evidenced by the appearance off colored spots. The spots were lightly
encircled with a pencil. The Rf values were calculated and compared.