Chem 31a- BIOCHEM LAB

Name: Hazelyn M. Benabaye Date Performed: November 19, 24

Group No: 6

CARBOHYDRATES

Experiment No. 3

Results and Discussion

Table1. Structural formulas for carbohydrates

MONOSA STRUCTURES
CCHARI FISCHER HAWORTH CHAIR
DES
glucose
 fructose

Table1.1 Disaccharides (Haworth and Chair structures)

Lactose
Haworth Chair

Maltose
Haworth Chair

Sucrose
Haworth Chair
POLYSACCHARIDES Amylose
Haworth Chair

Glycogen
Haworth Chair
Table1.2 Benedict’s test for reducing sugars

Compound Color Reducing Sugar (yes/no)

water blue No
glucose red-orange Yes
fructose red-orange Yes
sucrose blue-green No
lactose red-orange Yes
maltose reddish brown Yes
starch blue No
glycogen orange Yes

Benedict’s test for reducing sugar is a test that determines the amount and presence of
reducing sugar in a particular solution.

Sugars are classified as either reducing or non-reducing sugar based on their ability to act
as a reducing agent during Benedict’s test. The reagent in Benedict’s test for reducing sugar is
the Benedict’s solution which contains copper (II), sodium carbonate, and sodium citrate. The
copper solution gives a blue coloration. It reacts with the electrons from the ketone or aldehyde
group of the free reactive carbonyl group on the carbohydrate to form cuprous oxide (a red-
brown precipitate). This precipitate is formed due to the reduction of the Cu2+ ions to Cu+ ions,
In the process, the carbohydrate is oxidized. The color of the precipitate is dependent on the
volume of reducing sugar present. This means that the more free carbonyl groups present, the
increase in the amount of precipitate formed.
The reducing agent for reducing sugars is the aldehyde functional group (COOH), the
formyl group. Reducing sugars have either an aldehyde functional group or a ketone group in an
open chain form which is then converted into an aldehyde.

Reducing sugars are simple sugars and they include all monosaccharides and most
disaccharides. Examples of monosaccharides are glucose, fructose and galactose and examples
for reducing disaccharides are the lactose and maltose. Disaccharides sucrose is not a reducing
sugar. In fact, sucrose is the most common non-reducing sugar.,

Based on the Benedict’s test that was conducted, water and starch had no change in color.
Therefore, water and starch have no non-reducing sugars present. Glucose and fructose had its
change color of red-orange while the food sample which is the cake turned orange; this means
that a moderate amount of reducing sugar is present on the said compounds. On the other hand,
maltose had its color change of reddish brown; this means that a large amount of non-reducing
sugar was present. We didn’t obtain the accurate result for lactose, but lactose is a reducing
sugar.

Table 2.3 Barfoed’s test for monosaccharides

Compound Color Monosaccharides (yes/no)

water blue No
glucose light orange Yes
fructose red orange Yes
sucrose blue No
lactose blue No
maltose blue No
starch blue No
food sample (cake) blue No

Barfoed’s test is used to distinguish monosaccharides from disaccharides.

Monosaccharides are reducing sugars and they react faster with the reagent in order to form such
color change. Whereas, reducing disaccharides reacts slower than monosaccharides.

Based on the results that were obtain from the experiment, water, sucrose, lactose,
maltose, starch, and the food sample had the same color of blue upon heating. Only glucose and
fructose had a change in its color. Glucose turned into light orange while the fructose turned into
red orange. This means that only glucose and fructose were monosaccharides. Barfoed’s test
used copper (II) ions in a slightly acidic medium, reducing monosaccharides were oxidized by
the copper ion in solution in order to form carboxylic acid and an orange precipitate of copper
(Ii) within five minutes. Reducing disaccharides goes the same reaction but do so at a slower
rate. The non-reducing sugar gave a negative result. Barfoed’s reagent comprises of cupric and
acetic acid in solution. Monosaccharides readily react with the reagent in order to cause a
+
reduction in the 𝐶𝑢2 ions to 𝐶𝑢+ ions forming𝐶𝑢 2 𝑂. The reason behind this is that
monosaccharides, they oxidized readily in weak acid solutions. Disaccharides can as well reduce
the cupric ions; however, its reaction is much slower.

Table2.4 Seliwanoff’s test for ketohexoses

Compound Color Ketohexose/Aldohexose

water light orange Aldohexose
glucose light pink Aldohexose
fructose red Ketohexose
sucrose red Ketohexose
lactose light pink Aldohexose
maltose light pink Aldohexose
starch light pink Aldohexose
food product (cake) flesh colored Aldohexose

Seliwanoff’s test is used to distinguish between aldohexose and ketohexose. Seliwanoff’s

reagent consists of resorcinol crystals dissolved in equal amounts of water and hydrochloric acid.
Upon heating the solutions, the formation of a red precipitate indicates a positive result.

Based on the experiment, only fructose and sucrose had the positive result. They both
formed a red precipitate which indicated that it is a ketohexose. Other compounds had light
pink/light orange color; this indicates that they are aldohexose.

The acid hydrolysis of polysaccharides and oligosaccharides yields simple sugars. The
dehydrated ketose then reacts with the resorcinol in order to produce a red color. Aldose may
react slightly to produce a light pink color. Fructose and sucrose are two common sugars that
yield to a positive result. Sucrose is a disaccharide that consist fructose and glucose, that is why
it had a positive result. The test reagent dehydrated ketohexose in order to form 5-
hydroxymethylfurfural. Aldohexose reacts slowly much slower that ketohexose to give 5-
hydroxymethylfurfural, that is why it had only a light red color. Once the 5-
hydroxymethylfurfural was produced, it then reacts with resorcinol that gave a dark red
condensation product. Sucrose hydrolyzes to give fructose, which eventually reacted, that is why
it produced a dark red color.

Table 2.5 Iodine test for polysaccharides

Compound Color Polysaccharides (yes/no)

water yellow No
glucose light orange No
fructose light orange No
sucrose yellow No
lactose red orange No
maltose red orange No
starch black Yes (amylase)
food product (cake) black Yes (amylase)

Iodine test is used for the detection of starch in the solution. The black color is due to the
formation of starch-iodine complex. Starch contain polymer of α-amylose and amylopectin
which forms a complex with iodine to give the black color.

Based on the experiment, only the starch and the food product cake had the positive
result. They both formed into a black precipitate. Therefore, both the food product and starch are
polysaccharides. Other compounds had a negative result.
 Starch is composed of a straight chain subunit know as the amylase. Amylose is
responsible for the reaction with iodine. The amylase subunit of starch is made up of a straight
chain of alpha-glucose monomers that are connected by alpha 1á4 glycosidic bonds. In solution,
the amylase exists as a helically coiled structure. When the iodine was added, the molecules of
iodine became trapped within the helical structure and formed a complex one. Iodine has usually
a black color that causes the black coloration of starch which indicates that amylase is present.
The same with the food product cake, cake also result to a black precipitate because cake has an
ingredient of starch that had caused its black coloration.

Table 2.6 Hydrolysis of di- and polysaccharides

Compound Benedict’s Iodine

Sucrose(hydrolyzed) Yellow orange light yellow
Starch (hydrolyzed) blue black

This test is performed in order to breakdown to the monomer monosaccharide units. The
hydrolysis of the sucrose enabled the following results; in benedict’s test, the solution reduced
+
the 𝐶𝑢2 ions to 𝐶𝑢+ that resulted to the formation of the yellow orange precipitate. This means
that there was the presence of a reducing sugar in the solution, and since sucrose is not a
reducing sugar; the hydrolysis had created a monosaccharide that contains a free reactive
carbonyl group. In the Iodine’s test, the presence of amylase facilitated the reaction with the
reagent to form a black compound.

HCl breakdown the 1á2 glycosidic bond that links the glucose and fructose monomer
together to form sucrose. This break causes the release of two monosaccharides. Sodium
hydroxide was then added in order to neutralize the pH of the solution since the presence of acid
in the solution might disrupt the alkalinity of the benedict’s and iodine reagent that were added
after. Once it were hydrolyzed, the two reducing sugar were formed. This means that when
benedict’s test was performed, the solution was able to reduce the reagent and formed a yellow
orange precipitate. In Iodine’s test, the presence of amylase gave a positive indicat ion which
means that it is a polysaccharide.

On the case of starch, after adding a significant amount of hydrochloric acid to the starch
solution and heating the test tubes for a while, the iodine proved that a starch is a polysaccharide
and there is the presence of amylase because it formed into a black precipitate.

Once enough sodium hydroxide was added to the starch solution, it became slightly
basic. This experiment showed that starch can be rehydrated with HCl and heat. This breaks the
molecules up into single glucose molecules again causing the positive result in iodine’s test. H=
molecules break up the glucose molecules. However, because these have strong bonds, heat was
performed in order to create movement and weaken the bonds. This same process can also be
applied to sucrose in order to achieve a similar result, but this will happen much faster because
there are just enough bonds in sucrose compared to starch.

Compare the results of the Benedict’s test with sucrose before and after hydrolysis. Why are they
different?

Before hydrolysis, sucrose formed into a blue-green precipitate, whereas after hydrolysis it
formed into a yellow precipitate.

Sucrose is called a non-reducing sugar because it does not reduce copper sulphate, thus the
Benedict’s solution cannot break down the bonds of sucrose like other sugars. If sucrose is
hydrolyzed to its constituent monosaccharides (glucose and fructose), it will give a positive
Benedict's test. So sucrose is the only sugar that will give a negative Benedict's test before
hydrolysis and a positive test afterwards. Boiling the test solution with dilute hydrochloric acid
for a few minutes will hydrolyze the glycosidic bond forming the two monosaccharides or
(reducing sugars) that will now be capable of reacting with the Benedict's solution.
Compare the results of the Benedict’s test with starch before and after hydrolysis. Why are they
different?

Before hydrolysis, starch formed a blue precipitate, whereas after hydrolysis it had a different
color. Benedict’s reagent reacts with reducing sugars in order to produce a precipitate. Since
starch is not a reducing sugar, Benedict’s test had a negative result. However, the hydrolysis of
starch produces glucose which will react to Benedict’s reagent. As a result, benedict’s test after
hydrolysis had a positive result.

Compare the results of the Iodine test with starch before and after hydrolysis. Why is there a
difference?

Before and after hydrolysis on iodine’s test, both starch turned into a black precipitate which
indicates a positive result because of the presence of amylase.

Table 2.7 Fermentation test

Carbohydrates Height of the bubbles Fermented (yes/no)

Glucose 3.3 cm Yes
Fructose 2.1 cm Yes
Sucrose 2.0 cm Yes
Lactose 1.8 cm Yes
Maltose 1.4 cm Yes
Starch 6.5 cm Yes

Fermentation test using yeast is used to determine which carbohydrates substrates

positively influence yeast fermentation since yeast seems to have greater ability to utilize certain
carbohydrates.

Fermentation is a process that is important in anaerobic conditions when there is no

oxidative phosphorylation to maintain the production of ATP (Adenosine triphosphate) by
glycolysis. During fermentation pyruvate is metabolised to various different compounds.
Homolactic fermentation is the production of lactic acid from pyruvate; alcoholic fermentation is
the conversion of pyruvate into ethanol and carbon dioxide; and heterolactic fermentation is the
production of lactic acid as well as other acids and alcohols. Typical examples of fermentation
products are ethanol, lactic acid, and hydrogen. However, more exotic compounds can be
produced by fermentation, such as butyric acid and acetone. Although the final step of
fermentation (conversion of pyruvate to fermentation end-products) does not produce energy, it
is critical for an anaerobic cell since it regenerates nicotinamide adenine dinucleotide (NAD),
which is required for glycolysis. This is important for normal cellular function, as glycolysis is
the only source of ATP in anaerobic conditions.

Based on the results that were obtained from the experiment, all compounds were
fermented. On the height of bubbles of each of the compounds, glucose was 3.3 cm, fructose was
2.1 cm, sucrose was 2.0 cm, lactose was 1.8 cm, maltose was 1.4 cm and starch was 6.5 cm.

Table 2.8 Analysis of food products

Food sample Benedict’s Test Barfoed’s Test Seliwanoff’s Iodine Test

Test
biscuit + + - +
bread + + - +
rice + - - +
junk food + + - +
(piattos)
pork chop - - - +
cake + + - +

For the food samples that were brought by each group, the results on each tests had a
positive result and a negative result. The food samples biscuit, bread, and cake had a positive
result in Benedict’s, Barfoed’s, and Iodine test whereas on Seliwanoff’s test, it had a negative
result. On the other hand, the food sample rice had a positive result on both the Benedict’s test
and Iodine test but it had a negative result in both Barfoed’s and Seliwanoff’s test. The junk food
(Piattos) had a positive result on the three tests; the Benedict’s, Barfoed’s, and Iodine test but had
a negative result on Seliwanoff’s test. Pork chop had a negative effect in Barfoed’s, Benedict’s,
and Seliwanoff’s test but had a positive result on Iodine test.

Conclusion

Carbohydrates are the most abundant class of bioorganic molecules on planet earth.
Although their abundance in the human body is relatively low, carbohydrates constitute about
75% by mass of dry plant materials. Green (chlorophyll-containing) plants produce
carbohydrates via photosynthesis. In this process, carbon dioxide from the air and water from the
soil are the reactants, and sunlight absorbed by chlorophyll is the energy source.

There are several tests to use in order to determine a sample whether it is a carbohydrate
or not, if it is a monosaccharide or a disaccharide etc.

Some of the several tests use in determining carbohydrates includes, Benedict’s test for reducing
sugars, Barfoed’s test for monosaccharides, Seliwanoff’s test for ketohexose, Iodine test for
polysaccharides and hydrolysis of di- and polysaccharides.

Benedict’s test for reducing sugar is a test that determines the amount and presence of
reducing sugar in a particular solution. Barfoed’s test is used to distinguish monosaccharides
from disaccharides. Seliwanoff’s test is used to distinguish between aldohexose and ketohexose.
Iodine test is used for the detection of starch in the solution. Lastly, the hydrolysis of di- and
polysaccharides used to breakdown to the monomer monosaccharides.

Fermentation is also a test that can be used in carbohydrates. Fermentation test using
yeast is used to determine which carbohydrates substrates positively influence yeast
fermentation. Those following test that were stated above can help you determine the most
efficient methods for utilizing these test when dealing with carbohydrates.
References

http://www.scribd.com/doc/38760557/Characterization-of-Carbohydrates-Formal-Report#scribd

https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-1/

http://www.vvc.edu/academic/chemistry/10.%20Carbohydrates%20for%20colored%20-
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http://vlab.amrita.edu/?sub=3&brch=63&sim=631&cnt=1

http://www.nku.edu/~whitsonma/Bio150LSite/Lab%203%20Organic/Bio150LRevMolec.html