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Topic: Identification of unknown carbohydrate solution and investigation of action of saliva

and
Hydrochloric acid in carbohydrate solution at two different temperature
Objective: To identify the unknown carbohydrate solution and investigate effects of saliva
and HCL on carbohydrate solution at two different temperatures
Apparatus & Equipment:
Boiling tubes
Beaker
Metal test tube racks
Dropper
Water bath, ~37-40oC
Wooden holder
Water bath, ~90-95oC
Materials:
Carbohydrate solution A
Carbohydrate solution B
Benedicts solution 3 M

Hydrochloric acid 3 M
Sodium hydroxide

Procedures:
1. Prepare 4ml of NaOH, 18ml of Benedicts solution, 2ml of solution A, 10ml of solution
B, 8ml of HCl, dropper, 5ml measuring cylinder and metal test tube racks before
beginning the experiment
2. Prepare two boiling tubes containing 1 ml solution A and 1 ml solution B. Add 1 ml
Benedicts solution into each test tube. Both tubes are heated together in ~90-95oC
water bath for two minutes.
3. Add a few drops of solution A and B separately spaced on a white tile. On each
solution, add 1-2 drops of iodine. Mix them together on the tile with a glass rod.
Record observations
4. Pipette 2 ml solution B into each of four test-tubes and, label the tubes 1, 2, 3 and 4.
5. Place tubes 1 and 2 in a water bath of ~37oC.
6. 5ml of saliva are salivated into a boiling tube.
7. Pipette 2ml of saliva each into 1 and 4. Shake the contents of the tubes to ensure
thorough mixing.
8. Measure 4 ml HCl and pipette 2 ml each into tubes 2 and 3. Place tubes 3 and 4 in a
water bath set at ~95oC. Let tubes 1, 2, 3 & 4 incubate at their respective
temperatures for 35 minutes.
9. Label 4 more new tubes as 1, 2, 3 and 4. After 5 minutes of incubation of tubes
labelled 1 to 4 prepared previously, pour out 2ml of solution using measuring cylinder
from all these tubes into the respective newly labelled test tubes. Place back the
original tubes (labelled 1-4) back into the respective temperatures of incubation.
10. The acid in each of tube labelled 2 and 3 are neutralised with 1ml of sodium
hydroxide. Shake test tube 2 and 3 to ensure uniform mixing.
11. Carry out Benedicts test, 2ml of Benedicts solution are added to test tube 1 and 4,
3ml of Benedicts solution are added to test tube 2 and 3. Using a wooden holder,
shake and heat at 95C for one minute, shake continuously to prevent spitting.
Observations are recorded.
12. After 35 minutes of incubating tubes 1 to 4, neutralize the acid in each test tube
labelled 2 and 3 with 1ml of sodium hydroxide. 2ml of Benedicts solution are added
to test tube 1 and 4, 3ml of Benedicts solution are added to test tube 2 and 3. Heat
the solutions. Observations are recorded.
Results:
Table 1: Identification tests for solution A and B
Observations
Solution A
Benedicts test:
Iodine test:
Solution B
Benedicts test:
Iodine test:
Table 2:
Tube
1
2
3
4

Contents

Temp(oC)

2ml solution B
2ml saliva
2ml solution B
2ml 3 M HCl
2ml solution B
2ml 3 M HCl
2ml solution B

37
37
95
95

Conclusion

Benedicts Test-colour observation

After 5minutes
After 35minutes
(tube 1-4)
(tube 1-4)

2ml saliva

Discussion:
The enzyme involved in this experiment is salivary amylase. The enzyme hastens up the
time required to hydrolyse polymers into monomers. The enzymes act as a catalyst for
hydrolysis reaction and lowers the activation energy barrier required for the reaction to start.
Environment plays a major role in the hydrolysis process. Such as temperature and pH value
of the surrounding. In the experiment, salivary amylase is introduced to the carbohydrate
solution at two different temperatures, 37oc and 95oc.
Hydrochloric acid also acts as a catalyst for the hydrolysis of carbohydrates. (Organic
chemistry,) At 37oc with the presence of hydrochloric acid in solution B, the Benedicts test
result was negative, but at 95oc, the results returned for Benedicts test was positive. This
shows that even if hydrochloric acid catalyses the hydrolysis of carbohydrates, it doesnt
reduce the activation energy required for the reaction to start. Therefore, a higher
temperature was needed for the hydrolysis to happen.
Based on the result and observation obtained, the product obtained in boiling tube 1, 3 and 4
contain reducing sugar, same goes to boiling tube 1, 3, and 4 which also contain a greater
amount of reducing sugar due to a longer period of incubation.
At 37C, the mix solution of test tube 1 change colour from blue to yellowish-brown. If the
solution has a yellowish coloration, then a moderate amount of maltose is present (+++)
.After 35 minutes, the colour changed from yellowish to greenish-brown because the amount
of maltose increase. That was because the amylase enzyme in saliva speed up the process
of break down the starch into maltose. The mix solution of test tube 2 was remained the blue
colour because the temperature (37C) is too low and hydrochloric acid need more time to
break down the starch. So, we cant to observe the change in test tube 2 in a short time. At
95C, the mix solution of test tube 3 change colour from blue to pale blue after 5 minutes of
incubation because the pH of solution is acidic. Its mean if the more acid the solution, the
less the colour formed. After 35 minutes, the colour change to reddish brown because
hydrochloric acid speed up the process of breaking down the starch with the help of high
temperature. Then, the mix solution in test tube 4 also can change colour from blue to
yellowish-brown after 5 minutes of incubation in 95C water bath. That was because
temperature rises slowly and give the time for amylase to break down the starch. After 35
minutes, the temperature is approximately achieve boiling point and amylase was
denatured .So the colour of test tube 4 became dark brown.
The structure of the possible product obtained are as follow: Glucose Fructose Galactose
Reducing sugar of monosaccharide
Maltose Lactose

Reducing sugar of disaccharide

Sucrose Starch
Non-reducing sugar
Iodine test and Benedicts test are used in this experiment. For iodine test, the iodine is not
very soluble in water. Therefore, iodine reagent is more by dissolving iodine in water in the
presence of potassium iodide. Amylase in starch is responsible for the formation of deep
blue colour in the presence of iodine. Benedicts test detects the presence of reducing sugar.
The copper sulphate present in Benedicts test reacts with aldehyde or ketone group of the
reducing sugar to form copper oxide. Hence, a brown precipitate is formed, therefore the
colour given a point out of how much reducing sugar was present.
Carbohydrate in solution B is more complex compare to carbohydrate in solution A. Starch is
a polysaccharide