Objective: To investigate the action of saliva and 3M hydrochloric acid in two

carbohydrate solutions.
Apparatus & Equipment: Boiling tubes, Beaker, Graduated plastic dropper,
Water bath, ~37 ℃ , Water bath, ~95 ℃ .

Materials: Carbohydrate solution A, Carbohydrate solution B, Benedict’s

solution, 3 M Hydrochloric acid, 3 M Sodium hydroxide, Iodine solution
Procedures:
Part 1
1. Two boiling tube containing solution A and solution B were prepared. 1mL
of Benedict’s solution was added into each boiling tube. Both tubes were
heated together in (~95 ℃ ¿ water bath for two minutes. Results were
recorded in Table 1.
2. Few drops of solution A and solution B were added separately on a white
tile. 1-2 drops of iodine solution was added into both solutions. The
observation was recorded in table 1.
Part 2
3. Boiling tubes 1,2,3 and 4 were labeled. 2mL of solution B was pipetted into
each of four boiling tubes.

4. Boiling tube 1 and 2 were placed into ~37 ℃ water bath to heat up the
solution.
5. Saliva was collected in a small beaker till it reaches about 5mL.
6. This step was done approximately at the same time. 2mL of saliva was
pipetted into tubes 1 and 4. The content was shaken well to ensure thorough
mixing. 2mL of HCL was pipetted into tubes 2 and 3.
7. Tubes 1, 2, 3 and 4 were incubated for 30 minutes at their respective
temperature from this moment.
8. 4 more new boiling tubes were labeled tube 1’, 2’, 3’ and 4’.
9. After 5 minutes of incubation of tubes 1 to 4, 2mL of the contents from all
these tubes were poured out into the respective newly labeled tubes. The
original tubes were placed back into their respective temperature of
incubation.
10. 1mL of sodium hydroxide was added into tubes labeled 2’ and 3’ to
neutralize the acid. Tubes 2’ and 3’ were shaken to ensure uniform mixing .
11. Benedict’s test was performed on the contents of tubes 1’ to 4’ by
pipetting 2mL of Benedict’s solution into each tubes and heating them in 95
℃ water bath for 2 minutes. Observation was recorded in table 2.

12. After 30 minutes of incubating tubes 1 to 4, the acid in each test tube
labeled 2 and 3 was neutralized with 1mL of sodium hydroxide.
13. Benedict’s test was carried out for each tube with equal amount of
Benedict’s solution. The sample was heated. Observations were recorded in
table 2.

Flow chart for Part 2:

1 2 3 4

Solution B Solution B Solution B Solution B

2 ml 2 ml 2 ml 2 ml
Incubate at 37 ℃ 37 ℃ - -
Mix with 2 ml Saliva 2 ml HCL 2 ml HCL 2 ml Saliva
Incubate at 37 ℃ 37 ℃ 95 ℃ 95 ℃
After 5 minutes Remove tubes 1,2,3,4 from water bath
Pour out for 2 ml 2 ml 2 ml 2 ml
benedict's test
Pour to 1' 2' 3' 4'
Place tube 1,2,3,4 back into water bath for continuous
incubation
NaOH - 1 ml 1 ml -
Benedict's 2 ml 2 ml 2 ml 2 ml
solution
Heat for 1 min
Record observation at Table 12(After 5th min)
After 35 minutes Remove tube 1,2,3,4 from water bath.
Remaining 2 ml 2ml 2 ml 2 ml
content to
perform
Benedict's test
NaOH - 1 ml 1 ml -
Benedict's 2 ml 2ml 2 ml 2 ml
solution
Heat for 1 min
Record observation at table 2(After 35th min)
Results
(After 5 mins)
Results
(after 35 mins)
Table 1:

Observation Conclusions

Solution A Benedict's test: Presence of reducing sugar

Blue solution turns in solution A
into brick-red
precipitate

Iodine test:
Remains colourless

Solution B Benedict's test: Presence of starch

Solution colour in solution B
remains unchanged

Iodine test:
Colourless solution
turns into blue-black
colour

Tub Contents Temp ( Benedict's Test - Colour Observation

e ℃ ) After 5 min of After 30 min of
incubation incubation
(1'-4') (1-4)
1 2 ml 37 Blue solution colour Yellowish-green solution
solution B 2 turns to yellowish- colour with small
ml saliva green amounts of orange
precipitate

2 2 ml 37 Blue solution colour Blue solution colour

solution B 2 remains unchanged remains unchanged
ml HCL
3 2 ml 95 Little brick-red Colourless solution with
solution B 2 precipitate formed in formation of brick-red
 ml HCL blue solution precipitate

4 2 ml 95 Blue solution colour Blue solution colour with

solution B 2 turns to yellowish- formation of orange
ml saliva green precipitate

Discussions:
Discussion:
down
highly
Amylase
weak
enzyme
and
maltose
unit
has
and the
solutionspecific.
B
bonds
while
structure
reducing
insoluble
blue
brick-red.
the
being to
negative
B, isin
Amylase
won’t
complementary
hydrolyzes
into maltose
of
amaltose
complex
highly
Amylase
weak
enzyme
maltose
unit
blue more
Discussion:
down the
hydrolyzes
into be to
starch
and
the
structure
specific.
B
bonds
while
structure
reducing
insoluble
brick-red.
B, the
being
has ato
negative
complexa
solution is
of
in
A. in
Amylase
won’t
complementary be
starch
and
the toglucose
maltose
contains
in
amore
A. water.
This
maltose
contains
water.
This
structure itscontains
its substrate.
glucose at an
antwo
contains
substrate.
at Enzyme
optimum
twoEnzyme
optimum
simple Amylase
Amylase
sugar loses
temperature
simple temperature
sugar units.
units.
losesofits
ofits catalytic function.
95°C.
catalytic
95°C. function.

Enzyme Amylase was involved in the experiment. Amylase (enzyme) breaks

down the starch suspension (substrate), into maltose and glucose. The
reaction is highly specific.

Enzyme Amylase functions at an optimum temperature of 37°C. Therefore,

solution B in test tube 1 is broken down completely by the enzyme. However,
enzyme Amylase present in test tube 4 is denatured at 95°C. Due to high
temperature, the weak bonds holding the tertiary structure was broken down
and the 3D structure of enzyme Amylase was destroyed. The active sites of
enzyme Amylase were altered and won’t be complementary to its substrate.
Enzyme Amylase loses its catalytic function.

Hydrochloric acid functions as an inorganic catalyst for solution B. It

hydrolyzes starch into maltose and glucose at an optimum temperature of
95°C.

The products of the experiment are assumed to be maltose and glucose. Both
maltose and glucose are carbohydrates. Glucose is a monosaccharide while
maltose is disaccharide. The structure of glucose contains single simple sugar
unit while the structure of maltose contains two simple sugar units.

Benedict’s test was used to indicate the presence of sugar. Benedict's

solution contains copper sulphate ions (Cu2+), which are blue in colour. In the
presence of reducing sugar, copper sulphate ions were reduced to copper (I)
ions, which is insoluble in water. As a result, a brick-red precipitate is formed.
The solution colour turns from blue to brick-red.

The results of Benedict's test and iodine test for solution A is positive and
negative respectively, which concludes that solution A is a reducing sugar. For
solution B, the results are negative and positive respectively, which
concludes in solution B being a starch suspension. Therefore, solution B is
more complex compared to solution A. This is because starch is made up of a
large number of glucose units and has a more complex structure.