Liceo de Cagayan University

SY 2021-2022
First Semester

BIOCHEMISTRY

Name : Juliane Jayn A. Estoque Score: _______________

Course /Year & Section: 1st Year - BSN 1A Date: Sep. 10, 2022

Analysis of Carbohydrates
Laboratory Activity No. 2

I. LEARNING OBJECTIVES
1. Identify the chemical responses, reaction and properties that characterized a specific
kind of carbohydrates; and
2. Gain insight of the importance of carbohydrates in the series of biochemical reactions

II. CONCEPT NOTES:

A carbohydrate is an organic compound with the general formula Cn(H2O)n, that is,
consists only of carbon, hydrogen and oxygen. Carbohydrates make up the bulk of organic
substances on earth and perform numerous roles in living things. The carbohydrates
(saccharides) are divided into four chemical groups: monosaccharides, disaccharides,
oligosaccharides and polysaccharides. Polysaccharides serve for the storage of energy (e.g.,
starch in plants and glycogen in animals) and as structural components (e.g., cellulose in plants
and chitin in arthropods). Structural polysaccharides are frequently found in combination with
proteins (glycoproteins or mucoproteins) or lipids (lipopolysaccharides). The 5-carbon
monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD and NAD) and
the backbone of the genetic molecule known as RNA. The related deoxyribose is a component of
DNA. Saccharides and their derivatives include many other important biomolecules that play key
roles in the immune system, fertilization, preventing pathogenesis, blood clotting and
development.

III. TESTS ON CARBOHYDRATES

1) Molisch’s Test
Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds
containing carbohydrates in a combined form, based on the dehydration of the carbohydrate by
sulfuric acid to produce an aldehyde (either furfural or a derivative), which then condenses with
the phenolic structure resulting in a red or purple-colored compound.

See the video of the experiment/analysis. Click https://www.youtube.com/watch?v=etE8LCWvb-U

2) Carbohydrates as Reducing Sugars

A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group. The
enolization of sugars under alkaline conditions is an important consideration in reduction tests.
The ability of a sugar to reduce alkaline test reagents depends on the availability of an aldehyde
or keto group for reduction reactions. A number of sugars especially disaccharides or
polysaccharides have glycosidic linkages which involve bonding a carbohydrate (sugar) molecule
to another one, and hence there is no reducing group on the sugar; like in the case of sucrose,
glycogen, starch and dextrin. In the case of reducing sugars, the presence of alkali causes
extensive enolization especially at high pH and temperature. This leads to a higher susceptibility
to oxidation reactions than at neutral or acidic pH. These sugars, therefore, become potential
agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and so fort. Most commonly used tests for
detection of reducing sugars are Fehling’s Test, Benedict’s Test and Barfoed’s Test.

A. Fehlings Test
Fehling’s Solution (deep blue colored) is used to determine the presence of reducing
sugars and aldehydes.

See the video of the experiment/analysis. Click https://www.youtube.com/watch?v=yjLB_ntM324

 B. Benedict’s Test
Benedict’s solution is a deep-blue alkaline chemical reagent used to test for the
presence of the aldehyde functional group -CHO which consists of copper sulfate
pentahydrate (CuSO4. 5H2O), sodium carbonate (Na2CO3), sodium citrate (Na3C6H5O7) and
distilled water. Sodium carbonate renders alkaline conditions which are required for the
redox reaction, while sodium citrate is a complexing agent which complexes with the
copper (II) ions to avoid degradation into copper (I) ions during storage

See the video of the experiment/analysis. Click https://www.youtube.com/watch?v=TDFbtEwbmz0

C. Barfoed’s Test
Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so
that it can only be reduced by monosaccharides but not less powerful reducing sugars.
Disaccharides may also react with this reagent, but the reaction is much slower when
compared to monosaccharides.

See the video of the experiment/analysis. Click https://www.youtube.com/watch?

v=yQfMqvOxPrc

3) Seliwanoff’s Test

Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are
distinguished from aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone
group, it is a ketose and if it contains an aldehyde group, it is an aldose. This test is based on the
fact that, when heated, ketoses are more rapidly dehydrated than aldoses.

See the video of the experiment/analysis. Click https://www.youtube.com/watch?v=88hWF1-

RrTQ

4) Iodine Test
Iodine test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an
aqueous solution of potassium iodide) reacts with starch producing a blue-black color.

See the video of the experiment/analysis. Click https://www.youtube.com/watch?

time_continue=98&v=VeU7ULL9Usw&feature=emb_logo

IV. OBSERVATION
Write your observations from the Experiments/Analysis seen

TESTS ON OBSERVATION
CARBOHYDRATES

Molisch’s Test At the interface between the acid and the maltose reagent in this
test, I noticed a purple coloring that appeared as a ring, suggesting
the presence of carbohydrate. When the test method was performed
with the protein glycine, the surface began to turn green. Therefore,
glycine cannot be classified as a carbohydrate; giving a negative
result.

Fehling's Test In this test, I've come to the conclusion that a precipitate of copper
(1) oxide that displays various hues, as shown in the experiment,
signified a positive outcome.

Benedict’s Test The glycine, glucose, and maltose were tested, and were pronounced
as non-sugar, reducing sugar, and reducing sugar respectively.
Heating a mixture of Benedict’s qualitative reagent and a reducing
sugar such as glucose in a base medium, leads to the reduction of
 cupric ions in Benedict’s qualitative reagent to cupric ions; with the
formation of copper (1) oxide seen as a yellow to red precipitate in
the mixture.

Barfoed’s Test In this test, a formation of red or yellow to orange precipitation for
reducing monosaccharides yields a positive result.

Seliwanoff’s Test Seliwanoff's test allowed us to distinguish between aldose sugars

and keto-sugars; in the experiment, maltose, fructose, and glucose
produced aldose, ketoses, and aldose outcomes, respectively
(maltose; not red, fructose; cherry-red, and glucose; not red).

Iodine Test In this test, I observed that after applying iodine on the killed/dead
leaf, a blue stain appeared, indicating the presence of starch.

V. QUESTIONS/RESEARCH WORK

Molisch’s Test
1. Write the reaction step(s) involved in this test?
● Measure 2ml. of Maltose, a carbohydrate, into the test tube.
● Add 3 drops of Molisch reagent.
● Swirl to mix; and
● Run 2ml. of concentrated H2SO4 slowly along the inner walls of the test tube (Maltose +
Molisch reagent + Concentrated H2SO4).

Carbohydrates dehydrate in the presence of concentrated H2SO4— to yield furfural

derivatives. Furfural derivatives react with naphthol; a colored complex molecule (positive result).

2. Give an example of a protein structure that would give positive test with Molisch’s Reagent

Antibodies, a glycoprotein, is an example of a protein structure that would give a positive

test with Molisch’s Reagent. If a protein reacts positively in the Molisch test, the protein must
have covalently bound carbohydrate as part of its structure.

Fehling's Test
1. Write the reaction(s) involved in Fehling’s Test.

Copper ions are reduced from the oxidation state of +2 down to +1, which causes copper
(1) oxide to form and precipitate.Positive results in the Fehling test indicate presence of glucose,
fructose and lactose or presence of reducing sugar in the sample. The negative result of the
Fehling test indicates the presence of non-reducing sugars such as sucrose, starch.

2. What is the function of tartrate?

Fehling's solution is alkaline. Mixing cupric ions with an alkaline solution would
result in formation of cupric hydroxide of very low water solubility. By adding tartrate
ions, the cupric ions will form a complex with the cupric ions which will keep them
dissolved in the alkaline solution; forming a deep blue complex.
 3. Some disaccharides such as maltose are reducing agents, whereas others, such as sucrose
are not. Explain briefly by including the structures of the sugars.

Disaccharides such as maltose have an aldehyde group in its open chain form. That's why
this reaction can also be used to test for aldehydes. Sucrose on the other hand, the ring of the
glucose cannot open due to the glycosidic bond. Therefore, it is not a reducing sugar. But, it can
be splitted in acidic conditions. This way, all disaccharides can be splitted into monosaccharides;
giving a positive result.

Barfoed’s Test
1. Write the reaction(s) involved in the Barfoed’s Test.

A copper reduction test in which heating a mixture of a monosaccharide in a given time

interval results in the conversions with the formation of copper (1) oxide seen as a brick red
precipitate in the mixture. Unlike Benedict’s test, the copper reduction in Barfoed’s test occurs
in an acidic medium rather than in an alkaline medium.

2. When you test starch with Barfoed’s reagent, what would be the answer, positive or
negative? Explain your answer by giving reasons and structures.

Testing a starch with Barfoed’s reagent would yield a negative result. As mentioned, the
Barfoed's reagent is used to detect monosaccharides— starch is a polysaccharide consisting of
glucose monomers which are joined together with the help of 1,4 linkages. Hence, the result.

Seliwanoff’s Test
1. Write the reaction(s) involved in Seliwanoff’s Test.

When heated, ketoses are more rapidly dehydrated than aldoses, which explains why
aldoses only form a yellow to orange precipitate. Whereas keto dehydrates in the presence of
concentrated acids to yield furfural derivatives, which reacts with resorcinol in salivanos reagent
to yield a cherry-red complex; seen as a positive result.

2. What is the function of resorcinol?

Resorcinol works best for quick determination of keto-sugars because it produces a red
precipitate when combined with acid.

3. What is the aim of using a strong acid?

On treatment with concentrated/strong acid, ketoses are dehydrated more rapidly to give
furfural derivatives and on condensation than aldoses. Thus, I can say that strong acids are best
used for easy identification of keto-sugars and aldose-sugars, along with resorcinol.

4. What is the result of testing sucrose with Seliwanoff’s reagent? Explain your answers by
giving reasons and structures.

Seliwanoff's reagent would produce a positive result when used to test a sucrose. This is
due to the fact that sucrose, a disaccharide made of fructose and glucose, has a ketone group in
fructose, which interacts with resorcinol to produce a dark red (cherry) color.

Iodine Test
1. Explain the reaction between iodine solution and polysaccharides by giving the structures
of related compounds.

Polysaccharides can trap iodine in the center of their coiled and helical structures,
resulting in a blue-black color complex.

2. Each polysaccharide tested gives different color results with the iodine test. Explain the
reason briefly.
 The long chains are needed to make the reaction in an iodine test. Polysaccharides have
long carbon chains. Hence, the longer the chains, the darker the color complex it produces;
situated as “different color” results.