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, Benedicts
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 Benedicts 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. Benedicts test was performed on the contents of tubes 1 to 4 by
pipetting 2mL of Benedicts 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. Benedicts test was carried out for each tube with equal amount of
Benedicts solution. The sample was heated. Observations were recorded in
table 2.
Flow chart for Part 2:

Incubate at
Mix with
Incubate at
After 5 minutes
Pour out for
benedict's test
Pour to

NaOH
Benedict's
solution

After 35 minutes
Remaining
content to
perform
Benedict's test
NaOH
Benedict's
solution

Solution B
2 ml

Solution B
2 ml

Solution B
2 ml

Solution B
2 ml

2 ml HCL

2 ml Saliva

37

37

2 ml Saliva
37
2 ml

2 ml HCL
37

95

95

Remove tubes 1,2,3,4 from water bath

2 ml
2 ml
2 ml

1'
2'
3'
4'
Place tube 1,2,3,4 back into water bath for continuous
incubation
1 ml
1 ml
2 ml
2 ml
2 ml
2 ml
Heat for 1 min
Record observation at Table 12(After 5th min)
Remove tube 1,2,3,4 from water bath.
2 ml
2ml
2 ml
2 ml

2 ml

1 ml
2ml

1 ml
2 ml

2 ml

Heat for 1 min

Record observation at table 2(After 35th min)
Results
(After 5 mins)
Results
(after 35 mins)

Table 1:

Solution A

Observation

Conclusions

Benedict's test:
Blue solution turns
into brick-red
precipitate

Presence of reducing sugar

in solution A

Iodine test:
Remains colourless
Solution B

Benedict's test:
Solution colour
remains unchanged

Presence of starch
in solution B

Iodine test:
Colourless solution
turns into blue-black
colour

Tub
e

Contents

2 ml
solution B 2
ml saliva

37

2 ml
solution B 2
ml HCL
2 ml
solution B 2

37

Blue solution colour

remains unchanged

Blue solution colour

remains unchanged

95

Little brick-red
precipitate formed in

Colourless solution with

formation of brick-red

Temp (

Benedict's Test - Colour Observation

After 5 min of
After 30 min of
incubation
incubation
(1'-4')
(1-4)
Blue solution colour
Yellowish-green solution
turns to yellowishcolour with small
green
amounts of orange
precipitate

ml HCL
4

2 ml
solution B 2
ml saliva

95

blue solution

precipitate

Blue solution colour

turns to yellowishgreen

Blue solution colour with

formation of orange
precipitate

Discussions:
Discussion:
down
the
highly
specific.
solution
B
Amylase
weak
bonds
enzyme
Amylase
and
wont
be to
complementary
toglucose
itscontains
substrate.
Enzyme
Amylase
loses
its
catalytic function.
function.
hydrolyzes
into
maltose
starch
and
glucose
at an
antwo
optimum
temperature
ofits
95C.
maltose
isin
unit
while
the
structure
of
maltose
contains
two
simple temperature
sugar units.
units.
contains
reducing
insoluble
in
water.
blue
brick-red.
to
negative
B,
the
being
amore
A.
This
has
amaltose
more
complex
Discussion:
structure
down
the
highly
specific.
B
in
Amylase
weak
bonds
enzyme
Amylase
and
wont
be
complementary
its
substrate.
Enzyme
Amylase
loses
catalytic
hydrolyzes
into
starch
and
at
optimum
of
95C.
maltose
is
unit
while
the
structure
of
maltose
simple
sugar
contains
reducing
insoluble
in
water.
blue
brick-red.
to
negative
B,
the
being
a
solution
A.
This
has
a
complex
structure
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 37C. 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 95C. 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 wont 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
95C.
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.
Benedicts 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.