Activity 3
Activity 3
Activity 3
OBJECTIVE:
MATERIALS:
1 % of carbohydrate solutions( lactose, glucose, starch, sucrose, cellulose, fructose), distilled water (as
control tube), Barfoed’s reagent
APPARATUS:
Test tubes, test tube holder, 5 ml pipette, pipette filler, stop watch, water bath
CAUTION :
Barfoed’s reagent is corrosive and an irritant. If you spill any of the solution on yourself or on the bench,
immediately notify your laboratory instructor.
PROCEDURES
1. 5 ml of each of the carbohydrate solutions is added into one set of the labelled test tubes. 2. 5 ml of
Barfoed’s reagent is added to each test. 3. The contents of each tube are shook well. All the tubes are
placed in an actively boiling water bath at the same time. 4. After the water starts boiling again, the
solutions is heated for 3.5 min. ( Timing is important since a false positive test can be obtained for
monosaccharides with disaccharide, if the disaccharides are heated for more than 3.5 min thereby
breaking down ( hydrolyzing ) to monosaccharides ). 5. During this period, the tubes are observed closely
and any change of clarity of the solutions is noted. ( A positive test for monosaccharides is the
appearance of a red precipitate of Cu?O within 1 or 2 minutes, if no precipitate forms it indicates the
presence of a disaccharide).
RESULT:
1% CARBOHYDRATE SOLUTION
Fructose
Yes
Glucose
Yes
Cellulose
No
Lactose
No
Sucrose
No
Starch
No
No
DISCUSSION:
Barfoed’s test distinguishes monosaccharides from disaccharides. Positive test for monosaccharides is
the appearance of red precipitate (Cu2O) within 1-2 minutes. If no precipitate formed, indicates the
presence of disaccharide. The red precipitate come from the reaction between the reduction of copper
(II) acetate to copper(I) oxide (Cu2O). RCHO + 2Cu2+ + 2H2O > RCOOH + Cu2Ov + 4H+ The aldehyde
group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate.
Glucose and fructose which are monosaccharides show positive result in this test. Reducing
disaccharides undergo the same reaction, but do so at a slower rate. So, the timing to heat the sample is
set to 3.5 minutes. However, the samples are heated no more than 3.5 minutes to prevent the
disaccharide breaking down to monosaccharide. Lactose, sucrose, cellulose, starch and distilled water
showed negative result in this test.
CONCLUSION:
Only monosaccharide will give an immediate red precipitate in Barfoed’s test that is glucose and fructose
the other remaining solutions which are cellulose, lactose, sucrose, starch and distilled water do not
show any changes.
OBJECTIVE:
MATERIALS:
1% of carbohydrate solutions ( glucose, fructose, cellulose, lactose, sucrose, starch ), distilled water(as
control tube), Fehling solution A ( 69.28 grams copper (II) sulfate pentahydrate dissolved in 1 litre of
distilled water), Fehling solution B ( 346 grams Rochelle salt ( potassium sodium tartrate tetrahydrate)
and 120 grams sodium hydroxide in 1 litre of distilled water) APPARATUS:
5 ml pipette, test tubes, test tube holder, test tube rack, pipette filler, stop watch
PROCEDURES:
1. 5 ml of carbohydrate solutions is added into one set of test tubes. 2. By using different glass pipettes, 5
ml of Fehling A and 5 ml of Fehling B are added into each test tubes. 3. The solution is heated in a boiling
water bath for 5-10 minutes. 4. Red brick precipitate is formed for positive results.
RESULT:
Samples
Result
Lactose
Glucose
Fructose
Starch
Negative-no changes
Distilled water
Negative-no changes
Cellulose
Negative-no changes
Sucrose
Negative-no changes
DISCUSSION:
Fehling’s solution is used to test for the presence of a reducing sugar. Fehling’s solution was based on the
aldehyde or ketone groups in the sugar structures. A sugar is classified as a reducing sugar only if it has
an open-chain form with an aldehyde group or a free hemiacetal group. the presence of aldehydes but
not ketones is detected by reduction of the deep blue solution of copper(II) to a red precipitate of
insoluble copper oxide.
Fructose, glucose and lactose show positive result in this test. All monosaccharides are reducing sugars.
Many disaccharides, like lactose, also have a reducing form, as one of the two units may have an open-
chain form with an aldehyde group. However, sucrose, in which the anomeric carbons of the two units
are linked together, are non-reducing disaccharides since neither of the rings is capable of opening.
Polysaccharides (sugars with multiple chemical rings) are non-reducing sugars. Polysaccharides have
closed structures, which use free atoms to bond together their multiple rings, and take a much longer
time to be broken down. So, starch and cellulose which are polysaccharides have negative result in
Fehling’s test. Distilled water is not reducing sugar also shows negative result.
CONCLUSION:
Fehling test is the common test which is used to determine the presence of reducing sugar. Fructose,
lactose and glucose are reducing sugars which give brick red precipitate after the solutions are heated.
OBJECTIVE:
APPARATUS:
Test tubes, test tube holder, test tube rack, 5 ml pipette, pipette filler, dropper, stop watch, water bath
PROCEDURES
1. 5 ml of Benedict’s reagent and 2 ml of carbohydrate are added to a test tube and each tube is shook
thoroughly. 2. All the tubes are placed in a boiling water bath at the same time. The solutions are heated
for 5-6 min. 3. Any changes in color, in the transparencies and in the formation and color of any
precipitate are observed and recorded. 4. Later, 4 drops of 3M HCl are added to 5 ml of 1 % sucrose
solution and is heated in the boiling water bath for 5 min. 5. 1 % starch solution is treated in the same
way but the heating period was extended to 25-30 min. 6. 1-2 ml of each of solution is applied with
Benedict’s test in the same manner as before. 7. The results are compared with those obtained without
acid treatment.
RESULT:
Sugar solution
Starch
Light blue
Lactose
Sucrose
Light blue
Cellulose
Glucose
Distilled water
Light blue
Sucrose + HCI
Starch + HCI
Light
DISCUSSION:
The Benedict’s test is used to detect the presence of reducing sugars (sugars with a free aldehyde or
ketone group) such as glucose, fructose and lactose. All monosaccharides are reducing sugars; they all
have a free reactive carbonyl group. Some disaccharides have exposed carbonyl groups and are also
reducing sugars. Lactose which is disaccharides also called reducing sugar as it has the exposed carbonyl
groups. Other disaccharides such as sucrose and starch are non-reducing sugars and will not react with
Benedict’s solution. Benedict’s reagent is a mild oxidant with CuSO4, Cu (II) sulfate, as one of the
reagents. In the presence of a reducing sugar, the blue solution of Cu (II) or Cu+2, is changed to a brick
red/brown precipitate of Copper (I) or Cu+1 oxide,Cu2O. If there a small or large amount of the reducing
sugar present, the color would range from green to brick red respectively. RCHO + 2Cu2+ + 4OH- >
RCOOH + Cu2O + 2H2O Sucrose indirectly produces a positive result with Benedict’s reagent if heated
with dilute hydrochloric acid prior to the test, although after this treatment it is no longer sucrose. The
addition of HCl hydrolysed the non-reducing sugar, as it split it up into its component monomers.
The monomers are reducing sugars which gave the positive result on the second reducing sugar test. The
acidic conditions and heat break the glycosidic bond in sucrose through hydrolysis. The products of
sucrose decomposition are glucose and fructose, both of which can be detected by Benedict’s reagent,
as described above. This same goes for starch. But since starch has larger component compare to
sucrose so it took a longer time to hydrolyse. That the purpose of heat it in longer time compare to
sucrose. Without the addition of acid to sucrose solution, starch solution, the test given is negative. The
solutions remain clear blue after the addition of Benedict’s reagent and heating. Tap water is used only
to show the example of negative result of Benedict’s test. Thus it will not show any changes compare to
the carbohydrates.
CONCLUSION:
Benedict’s test is the common test which is used to determine the existence of reducing sugar. Fructose,
lactose, and glucose are reducing sugars which give positive test. Starch and sucrose are non reducing
sugars which give positive results after adding hydrochloric acid.
OBJECTIVE:
MATERIALS:
0.01M iodine, 0.12M KI , 1% carbohydrate solutions (cellulose and starch) , distilled water
APPARATUS:
PROCEDURE:
1. Few drop of 0.01M iodine in 0.12M KI added to 1% starch and cellulose solutions. 2. Any changes to
the colour are observed.
RESULT:
1% CARBOHYDRATE SOLUTION
COLOUR OBSERVED
Starch
Vivid blue
Cellulose
Yellowish brown
DISCUSSION:
Starch gives positive result in Iodine test as the color of solution change from yellow to dark blue. The
immediate formation of a vivid blue color indicates amylose. Vivid blue coloration forms due to the
polyiodide complex formed. Cellulose is derived from D-glucose units, which condensed through beta(1-
>4)-glycosidic bond. This give cellulose to be a straight polymer therefore, it can’t coil around iodine to
produce blue colour as starch does. Only starch gives the color of vivid blue, this is because it contains
amylase. The iodine molecules slip inside of the amylase coil. The amylose, or straight chain portion of
starch, forms helices where iodine molecules assemble, forming a dark blue color.
CONCLUSION:
The Iodine test is used to test for the presence of starch. Starch is a type of polysaccharide carbohydrate
which is made up of amylose and amylopectin. It is one of the main sources of carbohydrate and present
naturally in plant. Amylose in starch form dark blue complex with iodine.
SOLUBILITY TEST
OBJECTIVE:
MATERIALS:
Test tubes, test tube holder, glass rod, test tube rack, fume cupboard, 2 centrifuge tubes, analytical
balance, cylinder, graduated pipette, pipette filler, 2 evaporator dishes.
PROCEDURE:
1 5 g of starch is measured and put into a centrifuge tube; 2 40 ml of distilled of water is measured and
poured into the same centrifuge tube; 3 Step 1 to 2 is repeated by replacing the starch with cellulose; 4
Both of the tubes are heated: the tube containing starch is heated for about 2-3 minutes whereas the
tube containing cellulose is heated for about 10 minutes; 5 After heating, both of the content of the
tubes are allowed to cool down slightly; 6 The tubes are put into a centrifuge with 3500 rpm for 10
minutes; 7 Empty weight for both of the evaporator dishes is measured; 8 5 mL of the supernatant from
both of the tubes is pipetted and poured into two separate evaporator dishes; 9 The evaporator dishes
are left in the oven overnight
RESULT:
50.0023g
= 0.01860 %
50.0027g
= 0.002000 %
Carbohydrates
Weight of dry carbohydrates (gram)
Solubility (%)
Starch
50.0023g
0.01860
Cellulose
50.0027g
0.002000
DISCUSSION:
In this activity, the solubility is defined as the percentage ratio of the weight of dried supernatant to the
weight of the dry starch. Solubility can be interpreted as the amount of the dissolved compound that is
present in the test solution. From the calculations done, we can see that starch, with a percentage of
solubility at 0.01860 %, whereas cellulose have 0.002000 %. Starch and cellulose are two very similar
polymers. In fact, they are both made from the same monomer, glucose, and have the same glucose-
based repeat units. Since the sugar molecules contain the hydroxyl group or –OH, Thus it can form
hydrogen bonds with water molecules, which makes it soluble in water, but only to a limited extent.
However, the glucose units in starch are connected by alpha linkages while the glucose units in cellulose
are connected by beta linkages. In starch, all the glucose repeat units are oriented in the same direction.
But in cellulose, each succesive glucose unit is rotated 180 degrees around the axis of the polymer
backbone chain, relative to the last repeat unit. Although cellulose contains hydroxyl groups too, but
most of them are hydrogen-bonded to each other when the microfibrils stack together, which accounts
for the strength of cellulose fibers. There’s less free hydroxyl groups that can hydrogen bond with water
molecules, other than those hydroxyl groups that’s present at the end of each cellulose chain, which
causes the cellulose to be less soluble in water when compared to starch.
CONCLUSION:
Although both starch and cellulose are complex carbohydrates, which have large molecular weight size,
significantly reducing their affinity for water, but the hydroxyl groups that exist in the monomers itself
actually contributes to their insignificant solubility. However, the solubility will increase when these
complex carbohydrates are broken down into its monomers where the hydroxyl groups can form
hydrogen bonds with other water molecules easily due to the reduced molecular weight and size that
affects the affinity for water.
DIGESTIBILITY TEST
OBJECTIVE:
MATERIALS:
Starch powder, cellulose powder, enzyme amylase, benedict’s solution, distilled water
APPARATUS:
2 centrifuge tubes, measuring cylinder, analytical balance, pipette fillers, graduated pipettes, 2 droppers,
5 test tubes.
PROCEDURE:
1 5 g of starch is measured and put into a centrifuge tube; 2 40 ml of distilled of water is measured and
poured into the same centrifuge tube; 3 Step 1 to 2 is repeated by replacing the starch with cellulose; 4
Both of the tubes are heated: the tube containing starch is heated for about 2-3 minutes whereas the
tube containing cellulose is heated for about 10 minutes; 5 After heating, both of the tubes are allowed
to cool down slightly; 6 5 mL of starch is pipetted into a test tube; 7 Step 6 is repeated using a different
test tube but a drop of amylase is dropped into it; 8 5 mL of cellulose is pipetted into a test tube; 9 Step 8
is repeated using a different test tube but a drop of amylase is dropped into it; 10 5 mL of distilled water
is pipetted into the last test tube, and a drop of amylase is dropped into it; 11 20 drops of benedict’s
solution is dropped into five of the test tubes; 12 Any changes occurred is recorded and tabulated.
Result
Samples
Benedict’ s test
5 g of starch
blue
Negative
Positive
5 g of cellulose
blue
Negative
blue
Negative
Blue
Negative
DISCUSSION:
Amylase is one of the many members of a class of enzyme, hydrolases, that catalyze the hydrolysis of
starch into smaller carbohydrate molecules such as maltose (a molecule composed of two glucose
molecules). Two categories of amylases, denoted alpha and beta, differ in the way they attack the bonds
of the starch molecules. Alpha-amylase is widespread among living organisms. In the digestive systems
of humans and many other mammals, an alpha-amylase called ptyalin is produced by the salivary glands,
whereas pancreatic amylase is secreted by the pancreas into the small intestine. In the experiment, the
test tube that contains only distilled water served as a control for this experiment.
As for the test tubes that contain starch and cellulose without the amylase, they give a negative result for
Benedict’s test, because for starch and cellulose, since both of them are complex carbohydrates, thus
they have very few carbonyl groups which contribute to the compound’s reducing properties. Starch is a
non-reducing sugar which shows negative result in the Benedict’s test. As for the test tube that contains
starch and cellulose with the addition of a drop of enzyme, amylase, the test tube with starch gives a
positive result, but not the test tube with cellulose. As we all know, enzyme amylase can only catalyzes
the breakdown of starch into simpler sugars, but not cellulose. Cellulose only digested by cellulase
enzyme. It is impossible for human digestive enzymes to break the glycosidic bond. Therefore, only the
test tube containing starch treated with amylase gives a positive result for the Benedict’s test.
CONCLUSION:
Enzyme is a highly specific catalyst which can only converts a specific set of reactants into specific
products. Amylase only hydrolyze the starch but not cellulose. From here, we can say that the human
digestive system would not be able to digest the cellulose, because our digestive system only contains
amylase, and not cellulose. Therefore in the perspective of a human, we can conclude that the
digestibility of starch is higher than cellulose, provided that the enzyme amylase is present.
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