Title:

Identification of Unknown Carbohydrate Solution and Investigation of Action

of Saliva and Hydrochloric Acid in Carbohydrate Solution at Two Different
Temperatures.

Objective:

To identify unknown carbohydrate solution and investigate the action of

saliva and hydrochloric acid in carbohydrate solution at two different
temperatures which is 37C and 95C.

Apparatus:

Boiling tubes, Metal test tube racks, Water bath at 37C, Water bath at 95C,
Beaker, Dropper, Wooden holder, White tile and 10 ml measuring cylinder

Materials:

Carbohydrate solution A, Carbohydrate solution B, Benedicts Reagent, 3 M

Hydrochloric acid and 3 M Sodium hydroxide.

Procedure:
1. Two boiling tubes containing 1 ml of solution A and 1 ml of solution B is prepared.
1 ml of Benedicts solution is added into each boiling tube. Both tubes are heated
together in the 95C water bath for two minutes.
The results were recorded in Table 1.
2. Three drops of fresh solution A and B is separately dropped on a white tile. Two
drops of I2/KI solution (iodine) is added to each solution. A glass rod is used to mix
both solutions on the tile together. The observations is recorded in Table 1.
3. 2ml of solution B is added into four boiling tubes each using a pipette and the
boiling tubes is labelled as 1, 2, 3, and 4 respectively with a labelling paper near the
mouth of the tube. The initials of the group is written on the label.
4. Boiling tube 1 and 2 is placed into a 37 C water bath.
5. 5 ml of saliva is collected in a measuring cylinder.
6. 2ml of saliva is measured and added into boiling tubes 1 and 4 each using a pipette.
The test tubes are shaken well to ensure thoroughly mixing.
7. 2 ml of hydrochloric acid is measured using a pipette and added into boiling tube 2
which is already in the water bath and boiling tube 3. Boiling tube 3 and 4 is placed
in a water bath of 95C. The boiling tubes are let to incubate for 35 minutes in the
respective temperature. Boiling tubes 1 and 2 is in a 37C water bath while boiling
tube 3 and 4 is in 95C water bath.
8. Step 6 and 7 is done approximately the same time to ensure the time is constant.
9. Four new boiling tubes is labelled as 1, 2, 3 and 4. After 5 minutes of incubation of
tubes labelled 1 to 4 prepared previously, half of the total volume of the contents
from all the previous boiling tubes is poured into the respective new boiling tubes
(e.g., 1 into 1, 2 into 2 etc.). The original boiling tubes is immediately placed back
into the respective temperatures of incubation.
10. 1ml of sodium hydroxide is used to neutralize the acid in boiling tube labelled 2 and
3. (each). Boiling tubes 2 and 3 is shaken to ensure uniform mixing.
11. An equal volume of Benedicts solution which is 2 ml is added into each boiling
tube, to carry out Benedicts test. A wooden holder is used to shake and heat the
boiling tube at 95C or one minute. The boiling tubes is shaken continuously to
minimize spitting. All observations are recorded in Table 2.
12. The acid in boiling tube 2 and 3 is neutralized with 1 ml of sodium hydroxide after 35
minutes. Benedicts Test is carried out by removing 2ml of solution from boiling tube
1 to 4. 2ml of solution from each tube 1 to 4 is removed and Benedicts test with

an equal volume of Benedicts solution (2 ml) for each tube is carried out. The
sample is then heated at 95C. The observation is recorded in Table 2.
13. After 35 minutes of incubation for boiling tubes 1 to 4, the acid in the boiling tube
labelled 2 and 3 is neutralized. 2ml of solution from each boiling tube 1 to 4 and
Benedicts test is carried out with an equal volume of Benedicts solution (2ml) for
each tube. The samples are heated at 95C.
The observations are recorded in Table 2.
Results / Observation
Table 1: Test for absence and presence of the type of carbohydrates.

Solution A

Observations

Conclusions

Benedicts Test:
A brick red precipitate is formed in
a light solution.

Reducing sugar is present

in solution A.

Iodine Test:
A light yellow solution is formed.

Solution B

Starch is
solution A

absent

from

Benedicts Test:
The light blue solution remains the
same colour.

Reducing sugar is absent

from solution A.

Iodine Test:
A brownish
formed.

Starch is
solution B.

black

solution

is

present

in

Table 2: Test the effects of temperature and pH value to Solution B.

Tube

1
2

Contents

Temp
(C)

2 ml solution B
2 ml saliva
2 ml solution B
2ml
3M
Hydrochloric acid
2 ml solution B
2ml
3M
Hydrochloric acid

37

2 ml solution B
2 ml saliva

95

37

95

Benedicts TestColour Observation

After 5th min (from
tubes 1 4 into 1 4)

After 35th min (tubes

1 4)

A
greenish
blue
suspension is formed.
The clear blue solution
remains
the
same
colour.
The clear blue solution
turns to a greyish blue
solution with a brick
red precipitate.
A
light
green
suspension is formed.

A
dark
green
suspension is formed.
The light blue solution
gets lighter.
A dark blue solution
with
brick
red
precipitate is formed.
A
light
green
suspension is formed.

Discussion:

Carbohydrates are complex molecules which has to be broken down into simple soluble
molecules so that it can be absorbed by the body. This experiment studies the breaking
down of carbohydrates by manipulating the substances used and the temperature. Test I is
conducted to test the presence of carbohydrates and identify them. Test II is to test the
breaking down of starch by enzyme while manipulating the temperature. Test II also studies
the breakdown of carbohydrate by hydrochloric acid while manipulating the temperature.
In this experiment, the enzyme involved is salivary amylase that is found in the saliva.
Amylase is used because it is the enzyme involved in the digestion of carbohydrate.
Salivary amylase is secreted by the salivary glands in the mouth to start the breaking down
process of carbohydrate. Salivary amylase breaks down starch into maltose which is a
reducing sugar. By carrying out Benedicts test, we can determine the presence of maltose
in the sample because a brick red precipitate will be formed.
According to Test I, we have determined that Solution A is a reducing sugar. This is
concluded by the brick red precipitate formed by the solution when Benedicts test is carried
out. Test I also determines that Solution B is starch. This is due to the reddish brown iodine
solution turning blue black in the Iodine Test.
Starch is a polymer of glucose that is made of amylose and amylopectin. Amylose and
amylopectin molecules has 1,4 alpha-glycosidic linkage between carbon atom 1 of a
molecule and carbon atom 4 of another. Hydrochloric acid is added into test tube 2 and 3.
This introduces H+ ions into boiling tube 2 and 3. The hydrochloric acid acts as a catalyst
by speeding up the condensation process to break the glycosidic linkage. This
condensation process using hydrochloric acid needs heat to help the breaking down of the
1,4 alpha-glycosidic linkage. This is proven by the brick red precipitate that is formed in
boiling tube 3 proving that the starch has already been broken down into reducing sugar. In
boiling tube 2, the light blue solution remains the same due to the lack of heat to help the
acid to break the bond.
Saliva is used in test tube 1 and 4. Saliva contains the enzyme salivary amylase which
breaks down starch into maltose. Enzymes are highly specific. All reactions take place in
the active site of the enzyme which will only bind to the selected substrate with the right
shape and functional groups. This active site is affected by temperature because in high
temperature the active site will denature and the substrate will not be able to bind with the
active site. Salivary amylase works best at body temperature which is 37C. This is proven
by the dark green suspension formed in boiling tube 1 compared to light green suspension
in boiling tube 4.
In boiling tube 1, the dark green suspension leads to the conclusion that there is reducing
sugar in the boiling tube. The starch has been broken down to maltose by the salivary
amylase. The breaking down of starch into maltose has also happened I boiling tube 4
because of the light green suspension which indicates a small amount of reducing sugar
present. In boiling tube 2, the clear light blue solution remains the same because even
though the hydrochloric acid has been introduced into the solution, there is not enough heat
to aid in the breaking of the 1,4 alpha-glycosidic linkage. As mentioned earlier, solution B is
starch because it tested positive in the Iodines Test. Starch is a carbohydrate.

There are two test that has been carried out in this experiment which is Iodines Test and
Benedicts Test. Iodines Test is carried out to test the presence of starch in substance. If
there starch present, then reddish brown iodine turn blue black. Benedicts test is used to
test the presence of reducing sugar in a substance. The colour of the Benedicts test is
determined by the amount of reducing sugar in the substance. If none is present, then the
solution remains its clear blue colour. A green suspension is formed when there is small
amount of reducing sugar. This green colour gets darker as the amount increases and
eventually forms a brick red precipitate.
Carbohydrate A is a reducing sugar and Carbohydrate B is starch. Starch is a polymer of
carbohydrate which is made up of amylose and amylopectin. The reducing sugar is
predicted to be a monosaccharide or disaccharide. Starch is more complex compared to
the reducing sugar because amylose and amylopectin is highly branched with 1,4 alphaglycosidic linkage and 1,6 alpha-glycosidic linkage compared to the 1,4 alpha-glycosidic
linkage which is the linkage present in disaccharide. No glycosidic bonds are found in
monosaccharides.

Conclusion:
Saliva contains salivary amylase which will break down the carbohydrate into small simple
molecules. Temperature affects the enzyme. Enzyme works best at body temperature
(37C). Hydrochloric acid breaks down the glycosidic linkage of the carbohydrate but
requires heat to complete the breakdown of the glycosidic linkages.

Reference:

Books
Fosbery, Richard. (2009). OCR AS Biology Student Unit Guide: Unit F212 Molecules,
Biodiversity, Food and Health. London, United Kingdom : Hachette.

Websites
Santhosh, Lakshmi (Unknown) The Effects of Temperature on Enzyme Activity and Biology.
Retrieved from http://classroom.synonym.com/effects-temperature-enzyme-activity-biology6049.html