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Investigation of action of saliva and 3M hydrochloric acid in two carbohydrate solutions

Objective:

To study the relationship between two carbohydrates solution with saliva and
hydrochloric acid.

Apparatus:

Boiling tubes
Pipette filler
Water bath ( 37-40

Water bath ( 90-95

Beaker
Metal test tube racks
Graduated glass pipette ( 10ml )
Pasteur pipette
5ml Measuring cylinder
Labelling paper
Materials:

2ml of Carbohydrate solution A


42ml of Carbohydrate solution B
16ml of Benedicts solution
2ml of 3M hydrochloric acid
8ml of 3M sodium hydroxide
Distilled water
Small amount of iodine solution

Procedure:

First part
1) A few drop of solution A and B is added on a white tile separately.
2) 2 drops of iodine solution is added to mix with each solution by using a glass
rod.
3) The observation is recorded in Table 1.
Second part
1) 2ml of solution A and 2ml of solution B are added into two boiling tube each.
2) 2ml of Benedicts solution is added to each boiling tube.
3) Both boiling tube is heated in the water bath ( around 90-95
minutes.
4) The observation is record in Table 1.

) for two

5) 10ml of solution B is pipetted into each of four boiling tube. The boiling tube is
labelled with number 1, 2, 3 and 4 respectively with labelling paper near the
mouth of the test tube.
6) Boiling tube 1 and 2 is placed into water bath of ~37

and boiling tube 3

and 4 is placed into water of ~95 .


7) Saliva is collected with a height of 2cm in another test tube. Then the saliva is
diluted with 2cm of distilled water which is approximately equal volume with
saliva.
8) 2ml of diluted saliva is measured by using 5ml measuring cylinder and is
pipetted 1ml of the diluted saliva each into 2 empty test tube. Then the test
tube contained diluted saliva is poured into the boiling tube labelled 1 and 4.
The content inside the boiling tube is shook then placed back into water bath.
9) 2ml of hydrochloric acid is measured by using 5ml measuring cylinder and is
pipetted 1ml of hydrochloric acid into another 2 empty test tube. Then the test
tube contained hydrochloric acid is poured into the boiling tube labelled 2 and
3. The content inside the boiling is shook then placed back into the water
bath.
10) All of the boiling tube is set to incubate for 35minutes.
11) 4 new boiling tube is labelled with 1, 2, 3 and 4.
12) At the 5th minutes of incubation, boiling tube labelled 1, 2, 3 and 4 is poured
out about one-third of the total volume of the content into the newly labelled
boiling tube. Then the original boiling tube labelled 1, 2, 3 and 4 is put back
into the water bath.
13) The acid in the boiling tube 2 and 3 is neutralized with 2ml of sodium
hydroxide each.
14) 1ml of the solution in the boiling tube 1, 2, 3 and 4 is drawn out into new
boiling tube and labelled (1), (2), (3) and (4). 1ml of Benedicts solution for
each boiling tube is added. By using test tube holder, the newly labelled
boiling tube is heated in the water bath~95

for one minute. In this

process, the 4 boiling tubes is shook and the observation after one minute is
recorded in Table 2.
15) Boiling tube with label 1, 2, 3, 4, (1), (2), (3) and (4) is washed and 4 boiling
tube is labelled 1, 2, 3 and 4.
16) At the 35th minutes of incubation, boiling tube labelled 1, 2, 3 and 4 is poured
out about one-third of the total volume of the content into the newly labelled
boiling tube.
17) The acid in the boiling tube 2 and is neutralized with 2ml of sodium
hydroxide each.
18) 1ml of the solution in the boiling tube 1, 2, 3 and 4 is drawn out into new
boiling tube and labelled (1), (2), (3) and (4). 1ml of Benedicts solution for
each boiling tube is added. By using test tube holder, the newly labelled
boiling tube is heated in the water bath~95

for one minute. In this

process, the 4 boiling tube is shook and the observation after one minute is
recorded in Table 2.

Results:

Solution A

Observations
Benedicts test: brick-red solution is
formed
Iodine test: yellow solution is formed

Solution B

Benedicts test: blue solution is


formed
Iodine test: blue-black solution is
formed
Table 1

Temperature
Tube

Contents

10ml solution B
1ml saliva

37

10ml solution B
1ml 3M HCl

37

Conclusions
Presence of reducing sugar
No starch if presence
No reducing sugar is presence
Presence of starch

Benedicts Test Colour Observation


After 5th minutes
After 35th minutes
( from tube 1 4 into 1
( from tube 1 4 into
4 )
1 4 )
Blue solution turn to
Blue solution turn to
brick-red solution.
brick-red solution.
Brick-red precipitate is
Brick-red precipitate is
formed.
formed.
Blue colour is
remained.

Blur colour is
remained.

10ml solution B
1ml HCl

95

Blue solution turn to


yellow solution.

Blue solution turn to


brick-red solution.
Brick-red precipitate is
formed.

10ml solution B
1ml 3M saliva

95

Blue colour is
remained.

Blue colour solution


turn to pale blue
solution.

Table 2

Discussion:
This experiment contained 2 parts. The first part of the experiment is to test between
solution A and solution B which one is reducing sugar. The second part of the experiment is to
test and investigate the type of reactions. There are 2 types of reaction which is the biological
reaction and chemical reaction. Hydrochloric acid is the chemical enzyme and salivary amylase
is the biological enzyme.
There is an enzyme reaction in this experiment which is hydrolysis reaction. Solution B is
polysaccharide which will be break down into monosaccharide during the hydrolysis reaction.
Hydrochloric acid is strong acid, it will corrode or digest any object that in contact with it.
Hydrochloric acid will acts as chemical enzyme can hydrolyse polysaccharide at high
temperature. For solution B and starch, hydrochloride acid will break them down into their own
respective monosaccharide.
Temperature will have effect on saliva and may have effect on solution A and solution B.

saliva contains salivary amylase which need optimum temperature ~37

for maximum rate

of reaction. But high temperature will cause enzyme itself undergo denaturation. The shape of
the active site of the enzyme changes and causing from the bond between the enzymes itself
disturbed as heat energy is gained. As the shape changes the enzyme cannot function as usual,
thus no reaction can be taken out then the rate of reaction will drop or even does not occur. For
the case of solution B, at high temperature content in solution B gains heat thus increase the
kinetic energy, the activation energy can be achieve easily then it is easier to be catalysed by
the hydrochloric acid.
Products formed after the hydrolysis reaction is glucose as its been broken into
monosaccharide. This is because glucose is reducing sugar that will reduce the copper ions (

2+
+

Cu to become ( Cu which is from blue colour solution to get insoluble brick-red

precipitate in the solution. Thus as we can see in the observation as brick-red precipitate is
observed.

The structure of glucose as shown in the figure above.

The structure of starch as shown in the figure above.

The salivary amylase need to be in optimum temperature ~37

in order to carry out

the maximum reaction. In the other hand, the hydrochloric acid cannot carry out reaction in this
temperature as the temperature is not high enough to get that energy to catalyse the reaction.
But when in high temperature, hydrochloric acid will able to convert or break down starch to
glucose molecule because it gains enough energy from high temperature. But, salivary amylase
will denature at this high temperature. This condition will disturb the bond that hold the threedimension shape of the enzyme that altered the shape of the enzyme therefore the starch
molecule cannot bind with the denatured enzyme or active site altered enzyme. Benedicts
solution will not be success when in acidic condition. Therefore the alkaline is added before
running the Benedict test. The alkaline used is sodium hydroxide to neutralize the acidic
condition. Then the Benedicts solution is added to test the presence of reducing sugar for
example glucose.
Carbohydrate Solution B is the complex molecule so it will no show the positive result
of the reaction inBenedicts test. Before hydrochloric acid is added, solution B still is starch.
After hydrolysing reaction take place, is shows positive result to the Benedicts test that means
glucose is present in solution B after hydrolysing.
Conclusion:
Hydrolysis reaction involve salivary amylase enzyme can only take place when it is in

the optimum temperature condition ~37 . But will not show significant positive result in
high temperature condition~95 . Therefore, it will only show positive result in ~37
temperature condition. Hydrochloric acid required high temperature ~95

in order to

catalyse the reaction so it will only show positive result in the high temperature~95
condition. Both the result as shown in the Table 1 and Table 2 is same with the theory.

References:
Phil Bunyan. 2007. Two hydrolytic enzymes and an epistemologicalhistorical approach.
http://www.scienceinschool.org/2007/issue4/enzymes
http://brilliantbiologystudent.weebly.com/benedicts-test-for-reducing-sugars.html