Biochemistry class

1. • Activity no. 3
Prepared by: Group 2

2. OBJECTIVES
1. To be able to isolate polysaccharide from plant and animal tissue.
2. To identity of the isolated polysaccharide.

4. S
O
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5. 10 test tubes
graduatedcylinder
test tube brush
water bath
tripod
Bunsen burner
test tube holder
Spot plate
Crucible tong A
p
p
a
r
a
t
u
s

6. A
p
p
a
r
a
t
u
s
aspirator
pipette
watch glass
gauze beaker
Iron stand
funnel
Evaporatingdish
Mortar and pestle

7. ADDITIONAL MATERIALS:
CHEESE CLOTH
KNIFE
LITMUS PAPER
PLATFORM BALANCE
CHICKEN LIVER (50 GRAMS)
POTATOES (4 FIST SIZED)

8. I. ISOLATION OF POLYSACCHARIDE FROM:
A. CHICKEN LIVER
B. POTATO

9. A. POTATO
1. Wash and peel and grate 4 fist sized pieces of potatoes.
2. Fold the cheeseclothinto bags, then transfer the grated potatoes.
3. Place 100 mL of water in the beaker then soak and mash the cheeseclothwith grated
potatoes. Do this for about 5 mins. Notice the substancethat settles out.
4. Filter the solution using another cheesecloth.
5. Weigh 5.0 grams of residue and mix it with 20 mL of lukewarm water in a small beaker
and label this as solution A. Set this aside for part II.

10. B. CHICKEN LIVER
1. Rinse the liver with water, pat dry using the filter paper and weigh out 50 grams.
2. Cut the liver into small pieces and drop into 200 mL of boiling water.
3. Cover the container and continueboiling for about 20 mins. Or until the volume of the
solution should have been reduced about half
4. Note the transparency of the solution.
5. At the boiling point, faintly acidify with 10% acetic acid to coagulate the proteins.
6. Filter the solution while hot. Note the appearance of the filtrate.
7. Transfer the filtrate into test tube and label filtrate C. Set aside for part lI.

11. II. TESTS ON THE ISOLATED POLYSACCHARIDES
A. MOLISCH TEST
B. IODINE TEST

12. A. MOLISCH TEST
1. To 2 test tubes containing 2 mL of
solution A, & B respectively, add 2 drops of
Molisch reagent. Mix well.
2. Incline the test tube and carefully pour
down the side of the tube, 3 mL add
concentrated H2S04 such that it forms a
layer at the bottom.
3. Note the color at the interface of the 2
layers.
Solution A (Potato)Solution B (Liver)

13. Solution/
Filtrate
Molisch test
Solution A
(Potato)
The formation of blue-black product at the
interface of the two layers.
Solution B
(Chicken Liver)
the formation of a purple product at the
interface of the two layers.
A. MOLISCH TEST

14. ANALYSIS
Purple or violet ring confirms the presence of carbohydrate. Blue
color solution confirms the presence of starch.
Starch is present in potato because it reacted to iodine and
produced blue-black pigment. While the liver creates a yellow-
orange formation which is negative in the presence starch.

15. 3. IODINE TEST
1. Place a drop of iodine solution in an
evaporating dish, then add to it a drop of
fillrate A
2. Repeat the process with and solution B.
3. Note any color change in each solution
tested.

16. Solution/Filtrate Iodine test
Solution (A)
Potato
The formation of intense blue-black
product at the interface of the two
layers.
Solution (B)
Chicken liver
the formation of a yellow-orange
product at the interface of the two
layers.
3. IODINE TEST FOR POLYSACCHARIDES

17. ANALYSIS
Starch is present in potato because it reacted to
iodine and produced blue-black pigment. While the
liver creates a yellow-orange formation which is
negative in the presence starch.

18. II. HYDROLYSIS OF POLYSACCHARIDE

19. HYDROLYSIS OF POLYSACCHARIDE
1. Place 10 mL of solution A, B on two separate test tube and add 6 mL of 6N
HCI in a test tube. Immerse all tubes in a boiling water bath for 5 mins.
2. Test the solution by getting one drop of each sample and place it in
different depression on the spot plate. Add one drop of iodine solution.
Take note of the color of the reaction.
3. Continue testing/at one minute interval until the solution no longer
produces any change in color with iodine test solution.
4. Note all the color changes.
Color change with iodine test solution in the hydrolytic process.

20. Solution/
Filtrate
1st 2nd 3rd
Liver The liver filtrate
reacted with
iodine which
produced yellow-
orange color.
After a minute
of interval, a
drop of liver
filtrate and
iodine
produced
orange-yellow
color
After another
minute of
interval, liver
filtrate
produced
orange-brown
color
Potato produced black-
brown color.
potato filtrate
produced
reddish brown
and black
color.
potato filtrate
produced
reddish brown
color.

21. The results made a conclusion that as temperature
affects the color being produced by the solution.
When the solution is still high, color being produced
from the reaction of iodine and solutions A and B are
dark pigment for the potato filtrate and light for the
liver filtrate whereas the temperature lowers down,
the color of both solutions were starting to get the
same with iodine.

22. HYDROLYSIS OF POLYSACCHARIDE
5. Neutralize the solution by adding Na2CO3 test
solution to each tube. Check it using Litmus
paper.
6. Transfer 1 ml of each solution and place them
to three different test tube. Add 4 mL of
Benedict's reagent. Shake and place all in boiling
water bath. Note any precipitate form. Identify
the product formed after the hydrolysis.

23. QUESTIONS:
1. Why do we need to filter the acidified extract while if's hot?
The purpose of filtering the extract while hot is to filter the impurities
present while isolation. When you cool it down, acidified extract tends
to precipitate and then you'd be filtering oft product too.

24. The test in hydrolysis for
polysaccharides were unsuccessful.
The benedict’s test were not done
due to not lack of time and mislead of
procedure. The group should be
aware of time and the distribution of
task for equal and fast exhibition.