Title: Investigation of Action of Saliva and 3 M Hydrochloric Acid in Two

Carbohydrate Solutions
Objective: To investigate the action of saliva and 3M Hydrochloric Acid in Two
Carbohydrate Solutions
Apparatus & Equipments:
Boiling tubes

Metal test tube racks

Pipette filler

Graduated glass pipette, 10ml

Water bath, 37-40C

Water bath, 90-95C

Beaker

Pasteur pipette

Materials:
Carbohydrate solution A
Carbohydrate solution B
Benedict solution
3M Sodium hydroxide (or potassium hydroxide)
Procedure:
1. Two test tubes containing 2ml of solution A and 2ml of solution B is prepared. 2ml of
Benedict solution is added into each test tube. Both of the test tubes is heated together
in the hotter water bath of (90-95C) for two minutes. The results is recorded in table 1.
2. 10ml of solution B is pipetted into each of four test tubes and the tubes is labelled 1, 2, 3,
and 4 with labelling paper (or masking tape) near the mouth of test tube. The group
name is written on the labelling paper
3. Tubes 1&2 is placed in a water bath of 37C, and tubes 3&4 are placed in a water bath
of 95C (the duration of the test tubes in water bath doesnt matter because no saliva or
HCL added into the tubes)
4. 1cm 1.5cm of saliva is salivated into a test tube. The saliva is then diluted with an
approximately equal volume of distilled water
5. The addition of saliva or HCL into the respective tubes in step 6 is done at the same time
to ensure that the end product is consistent.
(the tubes that contain saliva in the water bath( 95C) shouldnt be left for more than
30s)
because enzymes denature when the the optimum temperature is exceeded.

6. 5ml measuring cylinder is used to measure out 2ml of diluted saliva prepared in step 3
and 1ml is pipetted into tube 1&4. The contents in the tubes is shaken well to ensure
even mixture.
7. 2ml of HCL is measured out using measuring cylinder and 1ml is pipetted into each
tubes 2(in water bath of 37C) and 3. Tubes 3 and 4 is placed in water bath set at 95C.
Tubes 1&2 ( in water bath of 37C), 3&4 (in water bath of 95C) is let to incubate at
their respective temperature. Table 2 is referred for 35 min from this moment.
8. 4 more (test tubes/ boiling tubes) is labelled as 1, 2, 3 and 4. After 5 minutes of
incubation of tubes 1-4, 1/3 of the content from the 1-4 tube is poured into the newly
labelled test tubes. (e.g., 1-1). The volume in the new test tubes is ensured at the same
level. This is to increase the consistency and the accuracy of the results as volume might
affect the end product. The test tubes of 1- 4 is placed back in the respective water bath
for incubation.
9. The acid in test tube labelled 2 and 3 is neutralized by adding 2ml of (sodium hydroxide/
potassium hydroxide) each. Test tube 2&3 is shaken to ensure even mixing.
10. 1ml of solution from tubes (1- 4) is transferred into new tubes and labelled so that
confusion did not take place. An equal volume of Benedict solution (1ml) is added to
each tube to perform Benedicts test. The tubes is shaken and heated at high
temperature for one minute (hotter water bath). Shaken continuously to minimize spitting.
The observation is recorded in table 2.
11. Test tubes 1- 4 is washed. After 35 minutes of incubating tubes1-4, about 1/3 of the total
volume from the test samples is poured into tubes labelled 1- 4.
12. The acid in test tubes labelled 2 and 3 is neutralized with 1ml of (sodium hydroxide/
potassium hydroxide). (Neutralization is used to ensure the acidity doesnt affect the
outcome of the experiment). 1ml of solution is removed from tubes 1- 4 and Benedicts
test is carried out with an equal amount of Benedicts solution (1ml) for each tube. The
sample is heated as mentioned in step 10.
13. Few drops of solution A and B is added separately spaced on a white tile. 1-2 drops of KI
solution is added to each solution and mixed with the pen cover. The observation is
recorded in Table 1.

Tabulation of data:

Table1: Identifying solution A and B

Observation
Benedicts test: Blue coloured solution

Solution A

Conclusion
Reducing sugar is present in

turns into brick red

solution A.

precipitate

Starch is absent in solution A

Iodine test: The blue solution remains

unchanged
Benedicts test: Blue solution

Solution B

Reducing sugar is absent in

remains unchanged
Iodine test: Black precipitate formed

solution B.
Starch is present in solution B

on dark blue solution

Table 2:
Tube

Contents

Temp (C)

10ml solution B

37

(frm tube1-4 into 1-4)

Blue coloured solution

(frm tube1-4 into1-4)

Brick red precipitate is

1ml saliva
10 ml solution B

37

turned into green

Blue cloudy solution

formed
Blue cloudy solution

1ml 3M HCI
10ml solution B

95

remains unchanged
White coloured

remains unchanged
The white coloured

translucent suspension is

translucent suspension

formed

turns into blue coloured

Blue coloured solution

translucent solution.
Blue colour solution

remains unchanged

remains unchanged

1ml 3M HCI

10ml solution B
1ml saliva

95

Benedicts Test-Colour Observation

After 5th min
After 35th min

Discussion:
The experiment was done to enhance the understanding regarding the action of enzyme on
the substrate. In this experiment the enzyme present was the salivary amylase and the
substrate is carbohydrate solutions. The amylase acts by breaking down starch which was
present in solution B according to the results obtained from Table 1. When HCI is added into

solution B, the amylase breaks down the starch into maltose and further breaking down
maltose into glucose. This happens because hydrolysis process has taken place in solution
B and the H+ ion present in the hydrolyzed solution B breaks the bond in between the
molecules in solution B.
Salivary amylase enzyme tends to work the best at its optimum temperature of 37C. This is
because all of these enzymes works in the body. Thus the optimum operating temperature is
body temperature of 35-40C. The solution B is broken down completely by the salivary
amylase enzyme. At the temperature of 95C which is above the optimum body temperature.
The molecules present in this enzyme will vibrate violently and the bond are broken. This
causes the structure and surface configurations of the enzyme is altered including the active
site which causes the enzyme to lose catalytic function. This enzyme is known to be
denatured. This is shown between the reaction between sugar and and Benedicts solution
which didnt take place due to denaturation of enzyme resulting in the solution to remain its
blue colour.
Benedicts solution is used to indicate the presence of sugar in the solution. Cu2+ present in
Benedicts solution is hydrolyzed by reducing sugar into Cu+ which is insoluble in water. The
colour change is from blue copper(II) ions to red-brown solution copper(I) ion. This results in
red precipitate solution formed in Table1.
The product of the experiment conducted in Table2 is predicted to be maltose and glucose.
Both of this glucose and maltose are carbohydrate. Glucose is an monosaccharide
consisting of a simple sugar unit but maltose is a disaccharide which consist of two simple
sugar unit( two units of glucose).
The result for Benedicts test for solution A is positive meanwhile for Iodine test its negative.
This shows solution A is a reducing sugar. The result for solution B for Benedicts solution is
negative meanwhile for iodine test its positive showing that solution B is starch. Starch has
more complex structure because it is made up of large number of glucose unit. It can be
concluded that solution B is more complex compared to solution A.
Conclusion: It can be concluded that solution A is recuing sugar and solution B is starch.